Method for determining a nucleic acid

ABSTRACT

New electron transfer moiety labeled nucleic acid analogue probes are provided that can be used in methods for determining nucleic acids in a sample. The new probes can be prepared using novel monomer subunits in a chemical synthesis route. The nucleic acids can be determined by binding the probe molecules to the nucleic acid and inducing electron transfer within the complex formed. The occurrence of the electron transfer is determined as a measure of the nucleic acid.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.08/805,411, filed Feb. 24, 1997.

The invention of this application was conceived with support from theUnited States Government, and specifically the National Institutes ofHealth.

FIELD OF THE INVENTION

The present invention is directed to methods for determining a nucleicacid in a sample with a probe molecule using electron transfer from anelectron donor to an electron acceptor. The invention is furtherdirected to compounds useful in such methods and to compounds useful forthe preparation of the probe molecules.

BACKGROUND OF THE INVENTION

Determinations of nucleic acids is becoming increasingly important as atool for diagnosis in the health fields. For example, the presence ofnucleic acids from organisms, like viruses, usually not present in thehuman body, can be determined using probes for the infecting nucleicacids. Further, any changes in the genome which may have a potentialinfluence on the metabolism and the state of health of the individualcan be determined. Such changes may have occurred by mutation or othermeans. Nucleic acid determination has made further progress with theintroduction of nucleic acid amplification procedures, like thepolymerase chain reaction (PCR).

The presently known nucleic acid assays can be divided into two types,the heterogeneous and the homogeneous assays. In heterogeneous assays,the nucleic acid is determined by binding to a nucleic acid probe whichis labeled for detection or by incorporation of labeled mononucleosidetriphosphates and subsequent immobilization of the so-labeled nucleicacid to a solid phase. This is preferably done by using a solid phasebound capture probe, a format which provides the advantage that anyexcess amount of labeled probes or mononucleotides can easily beseparated from the solid phase bound labeled nucleic acid. Thehomogeneous type of nucleic acid assay uses the interference between twolabels. In a first method, the two labels are linked together and theevent of hybridization initiates cleavage of the linkage between the twolabels. (The labels are chosen such that they elicit a signal as soon asthey are separated.) In a second method, the distance between the labelsis changed by hybridization events. In this case, the labels may belocated on one probe or on two separate probes having the capability ofhybridizing to the analyte nucleic acid such that the labels caninteract with each other.

Electron transfer between donors and acceptors is to be subdivided intotwo categories. In the first category, the donors (Do) and acceptors(Ac) are bound to the DNA duplex by non-covalent forces, such as van derWaals', electrostatic and hydrogen bonding. In the second class aresystems where Do and Ac are covalently linked to the DNA. The earliestdemonstration of the first approach was reported by Fromherz and Riegerin 1986, who studied photoinitated electron transfer (PET) fromintercalated ethidium to surface-associated methyl viologen (Fromherz,P.; Rieger, B. J. Am. Chem. Soc. 1986, 108, 5361). Electron transferproducts were demonstrated by direct observation of the reduced viologenacceptor. However, no special effect of the DNA, other than to provide ahigh effective concentration of the donor and acceptor, was observed. In1992, Harriman and Brun reported PET from ethidium and acridine donorsto diazapyrenium acceptors under conditions where the redox componentswere intercalated (Brun, A. M.; Harriman, A. J. Am. Chem. Soc. 1992,114, 3656). Multiexponential electron transfer kinetics were attributedto Do-Ac separations of 3, 4, and 5 base pairs. The B value derived inthat study (0.88 Å⁻¹) is comparable to that determined for Do-Ac systemsin proteins, where stacked π-electron systems are not available formediating electron transfer. Barton, Barbara and co-workers studiedtransition metal complex donors and acceptors which are intercalatedinto DNA and found that quenching of the donor fluorescence as well asrecovery of the ground state absorption proceeds at rates which areindependent of the number of bound acceptors, suggesting a very shallowdistance dependence for electron transfer through the DNA duplex (β<0.2Å⁻¹) (Arkin, M. R.; Stemp, E. D. A.; Holmlin, R. E.; Barton, J. K.;Hörmann, A.; Olson, E. J. C.; Barbara, P. F. Science 1996, 273, 475).However, cooperative binding of the donor and acceptor molecules, whichwould account for the loading-independent kinetics, could not becompletely ruled out in that system.

One of the problems associated with the use of non-covalently bounddonor and acceptor molecules in these studies is the inability tocontrol precisely the location of the redox components relative to oneanother when they are bound to the DNA. In one extreme, theintercalation locations will be controlled statistically, leading to adistribution of Do-Ac separation distances. At another extreme, bindingwill be cooperative, leading to short distances between Do and Ac over awide range of concentrations.

Covalent linkage of Do and Ac to the 5′-ends of complementaryoligonucleotides has led to systems with better defined Do-Ac separationdistances. Barton, Turro and co-workers reported fluorescence quenchingthat occurs in less than one nanosecond for a system containing linkedDo and Ac metal complexes intercalated near the ends of a 15 base pairduplex (Murphy, C. J.; Arkin, M. R.; Jenkins, Y.; Ghatlia, N. D.;Bossmann, S.; Turro, N. J.; Barton, J. K. Science 1993, 262, 1025). Arate this fast indicates that the distance dependence of electrontransfer through DNA is extremely shallow, but this interpretation mustbe regarded with caution pending a clear demonstration of redoxproducts. In contrast, there is a report of a covalently linked systemhaving Do and Ac metal complexes at the opposite ends of an 8 base pairduplex which shows electron transfer on a microsecond time scale (Meade,T. J.; Kayyem, J. F. Angew. Chem. Int. Ed. Engl. 1995, 34, 352). In thiscase the redox components were not intercalated within the helix so therate of electron transfer may simply reflect the time required to orientthe donor and acceptor in order to obtain sufficient electronic couplingthrough the π-electron stack before long distance electron transfer canoccur.

At this time, there are many unresolved questions regarding the abilityof duplex DNA to mediate electron transfer. None of the systems citedabove has unambiguously demonstrated the rate or efficiency of electrontransfer between donor and acceptor moieties held at a fixed distance ofseparation in a DNA/DNA duplex.

In a modification designed to detect hybridization of nucleic acids inhomogeneous solution, Tyagi and Kramer (Tyagi, S.; Kramer, F R. NatureBiotechnology 1996, 14, 303) describe a doubly substitutedsingle-stranded DNA construct that possesses a stem-loop (i.e. hairpin)structure. The construct contains a fluorescer covalently linked to oneterminus of the strand and an energy transfer quencher of the fluorescerat the opposite terminus. When unconjugated, this single stranded chainexists predominantly in hairpin conformation that constrains thefluorescer and quencher to be relatively close in space. When in thisstructural form, excitation of the fluorescer with actinic light leadsto reduced emission because the fluorescing excited state transfers itsenergy to the nearby quencher. However, when this single-strandedstructure hybridizes with a second strand complementary to its loopregion, the distance between the fluorescer and quencher is increasedand, consequently, the efficiency of fluorescence increases. The changein fluorescence intensity is an indicator that hybridization hasoccurred.

The modification described by Tyagi and Kramer offers several advantagesfor homogeneous real-time assays for hybridization. However there arecertain disadvantages to the system they report. First, the indicationof hybridization relies on energy transfer quenching of the fluorescer.This requires that the quencher have a lower excited singlet energy thanthe fluorescer, and this can cause difficulties in selecting a quencherwhose absorption spectrum does not overlap with that of the fluorescer.Second, the nature of the hairpin structure requires that a portion ofthe single-stranded probe molecule be self-complementary. In general,this self-complementary portion will not hybridize with the targetstrand of the nucleic acid to be determined. This requirement willreduce the association constant of the hybrid duplex DNA. A furtherdisadvantage of the modification described by Tyagi and Kramer is thatcovalent linkage of the fluorescer and quencher at the terminalpositions of the single-stranded DNA probe is cumbersome syntheticallyand far from ideal for an assay. The long chain of atoms used to bindthe fluorescer and donor to the single-stranded DNA is flexible and,consequently, the fluorescer and quencher will exist in manyconfirmations, even in the stem-loop structure, some of which may beineffective at quenching the emission of the fluorescer. This willcontribute to a high background fluorescence in assays forhybridization. Finally, a further disadvantage of covalent linkage ofthe fluorescer and quencher at the terminal positions is that unravelingof the stem structure at these positions, which is commonly to beexpected, will increase the distance between the fluorescer andquencher, and increase the number of available conformations. Both ofthese effects will lead to an increase in background emission.

In a further attempt to modify DNA Shimidzu and co-workers reported thesynthesis and characterization of a modified DNA oligomer containing anacridine moiety covalently linked at an internal position (Fukui, K.;Morimoto, M.; Segawa, H.; Tanaka, K.; Shimidzu, T. Bioconjugate Chem.1996, 7, 349). Hybridization with a complementary oligomer containingeither a thymine or an abasic site at the appropriate position oppositethe acridine yields a 1:1 duplex with the acridine, apparently,intercalated within the helix. Electron transfer to the acridine moietywas not reported.

Described in WO 95/15971 is the conjugation of oligonucleotides withintercalators that can act as electron donors or electron acceptors. Theresulting complexes represent a series of derivatives that arebimolecular templates whose use as probe molecules relies on duplex DNAto provide a path for the transfer of electrons over very largedistances at extremely fast rates. In this role the DNA duplex isdescribed as and must function as a “bioconductor”. In WO 95/15971 thereis disclosure of a method wherein oligonucleotides are labeled at eachend with different electron transfer moieties and it is demonstratedthat these moieties are capable of electron transfer through the duplexunder certain conditions. These electron transfer moieties are complexesof ruthenium and other heavy metal ions with organic ligands which canchange electronic state during electron transfer. Further in WO95/15971, there is a suggestion that the phosphodiester bonds in anoligonucleotide can be replaced by peptide bonds thus using peptidenucleic acids (PNA) as bioconductors.

SUMMARY OF THE INVENTION

The subject of the present invention is a method for determining anucleic acid in a sample comprising binding a probe having a polymericbackbone different from the natural sugar phosphate backbone of DNA orRNA to the nucleic acid by means of base-mediated hydrogen bonding,wherein this probe has an electron acceptor or an electron donor, orboth, bound covalently either at terminal positions or at internalpositions of the probe molecule. Further, stimulation of the electrondonor or electron acceptor by any one of several means elicits adifferent outcome depending upon whether the probe molecule is bound tothe nucleic acid. In particular, the present invention is a method forinducing an electron or hole transfer from an electron donor to thatelectron acceptor or a hole transfer from said electron acceptor to anelectron donor and determining the occurrence of the electronic transferas a measure of said nucleic acid. It is an object of the presentinvention to provide a method for reliable detection of electron or holetransfer.

A further object of the invention is to modify the efficiency or rate ofelectron (hole) transfer in bioinsulators and bioconductors.

In another aspect, the invention is directed to a method of controllingthe rate and efficiency of electron (hole) transfer with highspecificity for the indication of duplex formation.

From this prior art it is not apparent to one skilled in the art thatDNA/DNA, DNA/PNA and PNA/PNA hybrids will be bioconductors under allconditions of electron transfer driving force and time scale. Inparticular, electron conduction (electron transfer or hole transfer)from an electronically excited state, or other electron donor orelectron acceptor species not having an essentially infinite lifetime,must compete with return of the electronically excited state to itsground state, or consumption by some other means of the electron donoror acceptor with a limited lifetime. If conduction of an electronthrough a DNA/DNA, DNA/PNA and PNA/PNA duplex occurs on a time scalelonger than the return of the excited state to the ground state orconsumption of the electron donor or acceptor, then the DNA/DNA, DNA/PNAand PNA/PNA duplex will act as if it is a “bioinsulator”, rather than abioconductor.

The molecular and energetic features that control the rate andefficiency of electron transfer reactions have been extensively studied.The Marcus theory of electron transfer (Marcus, R. A. Ann. Rev. Phys.Chem. 1964, 15, 155. Marcus, R. A. J. Chem. Phys. 1965, 43, 679) isremarkably successful in its prediction of reaction rates. In itssimplest formulation (eqs. 1 and 2), the theory identifies three factorsthat determine the rate constant for electron transfer (k_(et)). Theseare the driving force (ΔG_(et)) for the reaction, the reorganizationenergy (λ), and the maximum rate constant (k_(max)), which occurs whenΔG_(≠)=0. In classical theory, k_(max)=k_(el)ν_(n), where k_(el) is theelectronic transmission coefficient and ν_(n) is the frequency ofpassage through the transition state. $\begin{matrix}{k_{et} = {k_{\max} \cdot {\exp \left( {{- \Delta}\quad {G^{\neq}/{RT}}} \right)}}} & (1) \\{{\Delta \quad G^{\neq}} = \frac{\left( {{\Delta \quad G_{et}} + \lambda} \right)^{2}}{4\quad \lambda}} & (2)\end{matrix}$

Of most relevance to the present invention is the estimation of k_(et)and its comparison with chemical or physical reactions that consume theelectronically excited state, or other electron donor or electronacceptor species not having an essentially infinite lifetime. If therate of electron transfer from donor to acceptor is greater than therates of the competing chemical or physical reactions, then the electrontransfer will be more efficient. Formation of a complex between theprobe and the nucleic acid to be determined can change the magnitude ofk_(et) and/or the magnitude of the rates of the chemical or physicalreactions that consume the electronically excited state, or otherelectron donor or electron acceptor species not having an essentiallyinfinite lifetime. Formation of the complex between the probe moleculeand the nucleic acid to be determined may modify ΔG_(et) so that therate of electron transfer increases or decreases upon complex formation.Alternatively, complex formation can cause changes in the environment ofthe electron donor or electron acceptor such that the rate of chemicalor physical reactions that consume the electronically excited state, orother electron donor or electron acceptor species not having anessentially infinite lifetime, changes relative to the rate for theelectron transfer reaction. Based on Marcus theory, the formation of thecomplex between the probe and the nucleic acid to be determined causes achange in the Marcus reorganization energy for the electron transfer ora change in the Marcus k_(max). A change in either parameter may resultin either an increase or decrease in the rate of electron transfer. Inthe case where formation of the complex between the probe and thenucleic acid to be determined results in an increase in the k_(et) theresulting complex can be called a bioconductor, since it facilitateselectron transfer. It is a surprising discovery that, depending on somespecific details of structure, formation of the complex between theprobe and the nucleic acid to be determined results in a decrease in therate constant for electron transfer in the complex. Thus, in some cases,the complex formed by the probe molecule and the nucleic acid to bedetermined may be a “bioinsulator”.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically the long range electron transfer (ET)mechanism of the present invention. The concept is illustrated by theexample where the donor and acceptor are located on the same strand. Thedesignations n and m refer to the electronic states of the donor and theacceptor.

FIG. 2 shows a first embodiment of the invention using only one probemolecule labeled with a distally positioned electron donor (Donor) andan internally positioned acceptor (acc.).

FIG. 3 shows an embodiment of the present invention wherein two labeledprobes are used. Thus one probe contains the acceptor (distally located)and another probe contains the donor (distally located).

FIG. 4 shows a format using an immobilized probe containing aninternally positioned acceptor and donor in the same probe.

FIG. 5 shows the proposed electron transfer mechanism via hole hopping.

FIG. 6 shows a format using a hairpin forming probe.

FIG. 7 shows an embodiment using two complementary probes.

FIG. 8 shows the flow scheme for the preparation of Q1, an electronaccepting PNA monomer (linker length 1).

FIG. 9 shows the flow scheme for the preparation of Q2, an electronaccepting PNA monomer (linker length 4).

FIG. 10 shows the flow scheme for the preparation of R1, an electrondonating PNA monomer (linker length 4).

FIG. 11 shows the flow scheme for the preparation of R2, an electrondonating PNA monomer (linker length 7).

FIG. 12 shows absorbency/temperature profiles for PNA/DNA hybrids withdifferent quinone linker lengths for different wavelengths. AQ₂ standsfor PNA579 and AQ₅ for PNA586.

FIG. 13 shows photoinduced cleavage of DNA within PNA/DNA hybrids.

FIG. 14 shows photoinduced cleavage of DNA with an 8-OxoG site.

FIG. 15 shows phosphorescence quenching in PNA/DNA hybrids due toelectron transfer.

FIG. 16 shows absorbency/temperature profiles for PNA626 and 627hairpins.

FIG. 17 shows absorbency/temperature profiles for DNA626A hybrids withPNA626 and PNA627.

FIG. 18 shows schematically the synthesis of a further PNA-electrondonor monomer.

DATAILED DESCRIPTION

The term nucleic acid to be determined is understood in the presentinvention as an analyte nucleic acid or a nucleic acid derivedtherefrom. Analyte nucleic acids include nucleic acids of any origin,for example, nucleic acids of animal, human, viral, bacterial, orcellular origin. They may be present in solution, suspension, but alsofixed to solid bodies or contained in cell-containing media, cellsmears, fixed cells, tissues, or fixed organisms. Nucleic acids derivedtherefrom are nucleic acids prepared from analyte nucleic acids or partsthereof, for example as copies of the above-mentioned nucleic acids, orparts thereof. These copies include nucleic acids derived from thatoriginal analyte nucleic acids by amplification, including anyreplication or/and transcription/reverse transcription reactions, forexample by the polymerase chain reaction.

Usually the nucleic acid to be determined will be pretreated to get intoa condition ready for binding the probe molecule, if it is not alreadyaccessible. Such pretreatment may include denaturing double-strandednucleic acids by changing the pH into the alkaline region, repeatingextreme temperature changes (freezing/thawing), changing thephysiological probe conditions (osmotic pressure), lysis of cell wallsby detergents, chaotropic salts or enzymes (e.g. proteases, lipase).These steps may be used either alone or in combination in order torelease the nucleic acids. In some instances, it may be advantageous toseparate the nucleic acids from other components of the sample, likeproteins, cells, cell fragments, but also nucleic acids which are notintended to be detected.

The probe molecule binding to the nucleic acid is defined by having apolymeric backbone different from the natural sugar phosphate backbone.Examples of such probe molecules are now well-known in the art. Thoseprobes may be based upon monomeric subunits, which are linked in arepetitive way. It is preferable that the probe molecule contains atleast five monomeric subunits, each monomeric subunit being connected toother monomeric subunits by peptide bonds. The peptide bond isunderstood to be the bond connecting a primary or secondary amine and acarboxylic acid residue. Other types of linkages within the monomers orconnecting one or more monomers in the backbone are possible, forexample, ether or amino bonds. Examples of such probe molecules aredescribed in WO 92/20702 including probe molecules having ligands boundto aza-nitrogen atoms, WO 94/25477, and WO 96/20212. Probe moleculeshaving mixed different linkages between the monomers are described inEP-A-0 672 677. The term probe molecule further contains moleculeshaving the above stretch of the non-natural backbone and an additionalstretch of the natural sugar phosphate backbone. Such chimera maypossess some lower affinity to complementary nucleic acids, but arenevertheless useful in the present invention.

The term backbone in the present invention shall mean the polymericmoiety to which at different points of attachment heterocyclic basemoieties are bound in a consecutive way.

The binding of the probe molecule to the nucleic acid is accomplished byhydrogen bonds mediated by said base moieties on said backbone. Suchhydrogen bonding for example occurs between complementary nucleobases asin the base pairing in nucleic acids having the natural sugar phosphatebackbone or artificial bases eliciting similar properties for hydrogenbonding to the natural bases. While the most prominent mode of bindingis by duplex formation, some molecules bind to nucleic acids by triplexformation (see for example WO 95/01370). Artificial bases includediaminopurine, pseudouridine, thioguanine and 7-deazaguanine. The methodof the invention requires a stretch of at least five consecutivemonomeric units coupling the bases of the probe molecule. The probemolecule should have a length of at least 9 base-pairing monomericunits, preferably between 9 and 30 and most preferably between 10 and 25base-pairing monomeric units.

Such probe molecules can be prepared by analogy to the methods describedin the above-mentioned documents. Their preparation may includetechniques usually employed in peptide chemistry. However, probemolecules especially preferred in the present method include newstarting and intermediate compounds useful for introducing any electrondonor or/and any electron acceptor moieties into the probe. Theirpreparation will be described later.

An electron acceptor or an electron donor or an electron acceptor and anelectron donor are bound covalently to the probe molecule at defined,fixed positions, according to the invention. The generally possiblepositions for attaching moieties to polymeric backbones can besystematically divided into a first group wherein the moiety is attachedat one or both ends of the polymeric backbone, a second group wherein atleast one of the groups is bound to the polymeric backbone at a positionon the backbone not located on the first and the last base bearingmonomeric unit of said backbone, and a third group wherein the moiety isbound to a base position attached at any of the monomeric units.Surprisingly, the objects of the present invention are better met forthe second group. Within this group, such probe molecules have proven tobe the most successful in which the nucleobase moiety of one or more ofthe monomeric subunits is replaced totally by a group or groupscontaining the electron acceptor or an electron donor, preferably suchthat the rate or efficiency of electron transfer in the probe ismodified by the nucleobases of the nucleic acid to be determined. It ispossible not only to modify only one subunit by such moieties, but alsomore subunits, dependent upon the overall length of the probe molecule.However, as a general rule, it is preferred to not include more than 30%of modified monomeric units compared to the overall number of monomericunits in the probe molecule. If more than one modified monomeric subunitis contained within the probe molecule, these monomeric units should notall be located at the final monomeric subunits.

Preferred probe molecules are compounds of the general formula I

wherein

n is an integer of from at least 3,

x is an integer of from 2 to n−1,

each of L¹-L^(n) is a ligand independently selected from the groupconsisting of hydrogen, hydroxyl, (C₁-C₄)alkanoyl, naturally occurringnucleobases, non-naturally occurring nucleobases, aromatic moieties, DNAintercalators, nucleobase-binding groups, heterocyclic moieties,reporter ligands and chelating moieties, wherein at least one ofL¹-L^(n), preferably at least one of L²-L^(n−1) is a non-nucleobaseelectron acceptor or a donor moiety and at least 2 of L¹-L^(n) being anucleobase binding group, or a naturally or non-naturally occurringnucleobase;

each of C¹-C^(n) is (CR⁶R⁷)_(y) (preferably CR⁶R⁷, CHR⁶CHR⁷ or CR⁶R⁷CH₂)where R⁶ is hydrogen and R⁷ is selected from the group consisting of theside chains of naturally occurring alpha amino acids, or R⁶ and R⁷ areindependently selected from the group consisting of hydrogen,(C₁-C₆)alkyl, aryl, aralkyl, heteroaryl, hydroxyl, (C₁-C₆)alkoxy,(C₁-C₆)alkylthio, NR³R⁴ and SR⁵, where R³ and R⁴ are as defined below,and R⁵ is hydrogen, (C₁-C₆)alkyl, hydroxyl, (C₁-C₆)alkoxy, or(C₁-C₆)alkylthio-substituted (C₁-C₆)alkyl or R⁶ and R⁷ taken togethercomplete an alicyclic or heterocyclic system; or C¹-C^(n) is CO, CS,CNR³;

each of D¹-D^(n) is (CR⁶R⁷)_(z) (preferably CR⁶R⁷, CHR⁶CHR⁷ or CH₂CR⁶R⁷)where R⁶ and R⁷ are as defined above;

each of y and z is zero or an integer from 1 to 10, the sum y+z being atleast 2, preferably greater than 2, but not more than 10;

each of G¹-G^(n−1) is —NR³CO—, —NR³CS—, —NR³SO— or —NR³SO₂—, in eitherorientation, where R³ is as defined below;

each of A¹-A^(n) and B¹-B^(n) are selected such that:

(a) A¹-A^(n) is a group of formula (I/A), (I/B), (I/C) or (I/D), andB¹-B^(n) is N or R³N⁺; or

(b) A¹-A^(n) is a group of formula (I/D) and B¹-B^(n) is CH;

 wherein:

X is O, S, Se, NR³, CH₂ or C(CH₃)₂;

Y is a single bond, O, S or NR⁴;

each of p and q is zero or an integer from 1 to 5, (the sum p+q beingpreferably not more than 5);

each of r and s is zero or an integer from 1 to 5, (the sum r+s beingpreferably not more than 5);

each R¹ and R² is independently selected from the group consisting ofhydrogen, (C₁-C₄)alkyl which may be hydroxyl- or (C₁-C₄)alkoxy- or(C₁-C₄)alkylthio-substituted, hydroxyl, (C₁-C₄)alkoxy, (C₁-C₄)alkylthio,amino and halogen; and

each R³ and R⁴ is independently selected from the group consisting ofhydrogen, (C₁-C₄)alkyl, hydroxyl- or alkoxy- or alkylthio-substituted(C₁-C₄)alkyl, hydroxyl, (C₁-C₆)-alkoxy, (C₁-C₆)-alkylthio and amino;

Q and I is independently selected from —CO₂H, —CONR′R″, —SO₃H or—SO₂—NR′R″ or an activated derivative of —CO₂H or —SO₃H and —NR′R′″

where R′, R″ and R′″ are independently selected from the groupconsisting of hydrogen, alkyl, amino protecting groups, reporterligands, intercalators, chelators, peptides, proteins, carbohydrates,lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates,nucleotide triphosphates, oligonucleotides, including botholigoribonucleotides and oligodeoxyribo-nucleotides, oligonucleosidesand soluble and non-soluble polymers and as well as nucleic acid bindingmoieties and

each of x1 and y1 is an integer offrom 0 to 10.

Alkoxy- and alkylthio groups contain preferably from 1 to 4 carbonatoms.

An example for a fused aromatic moiety is naphthol. A heterocyclicmoiety is pyridine. Reporter groups are moieties that can be detected,like fluorescent compounds, for example fluorescein, or moieties thatcan be recognized by another molecular entity, like haptens which can berecognized by an antibody raised against this hapten.

In the above structures wherein Q or I is an oligonucleotide oroligonucleoside, such structures can be considered chimeric structuresbetween PNA compounds and the oligonucleotide or oligonucleoside.

Linkers A¹-A^(n) for binding acceptor moieties are generally preferredat a length of between 1 to 10 atoms, most preferred 2 to 6 atoms, fordonor moieties at a length of 1 to 10, most preferred 2 to 8 atoms.

More preferable are compounds of subgroups Ia-Ib based on the generalformula I wherein

(Ia): B¹-B^(n) is N and A¹-A^(n) is —CO—(CH₂)₆—

(Ib): B¹-B^(n) is N and A¹-A^(n) is —CO—NR³—(CH₂)₂—

(Ic): B¹-B^(n) is CH and A¹-A^(n) is —NR³—CO—(CH₂)₂—.

Preferred PNA-containing compounds useful to effect binding to RNA,ssDNA and dsDNA and to form triplexing structures are compounds of theformula IIa, IIb and IIc:

wherein:

each L is independently selected from the definitions of L¹-L^(n) informula I;

each R⁷ is independently selected from the group consisting of hydrogenand the side chains of naturally occurring alpha amino acids;

n is an integer greater than 1,

each k, 1, and m is, independently, zero or an integer from 1 to 5;

each p is zero or 1;

R^(h) and R^(i) are as defined for R′, R″ and R′″.

Electron acceptor moieties and electron donor moieties are each referredto as electron transfer moieties. They are most preferably moieties notincluding nucleobases (non nucleobase moieties).

Electron acceptors according to the present invention can induceoxidation of other moieties by attracting an electron from theirsurroundings particularly after they have been stimulated with light oractivated by some other means. Electron acceptors will typically beelectron-deficient species. Preferable electron acceptors are organicmolecules, preferably excluding organic molecules containing metal ions.Further, these organic molecules have a generally flat structure with asystem of delocalized π-electrons. Therefore, organic molecules areespecially preferred that contain aromatic hydrocarbon moietiescontaining functional groups, wherein the functional groups may haveelectron-withdrawing properties. It is possible that the electronacceptor will contain both electron-donating and electron-withdrawingfunctional groups. The substituents should not be so bulky that theydisturb the regular base stacking between the probe and the nucleic acidto be determined. The electron deficiency of the electron acceptor willnormally be increased by excitation with light or with stimulation bysome other means. In the preferred case there is no measurable electrontransfer to the acceptor without its stimulation in the assay medium.The acceptor moiety will typically have well-characterized redox andspectroscopic properties, although precise knowledge of these parametersis not required for their successful application in this invention.Electron acceptors useful in this invention typically undergo a changein chemical or optical properties when it accepts an electron. Forexample, such electron acceptors may be useful in this invention when,after conversion by electron attraction to become a radical anion, orany other one electron reduction product, it possesses a unique opticalspectrum with strong absorption bands. However, the detection of thechange in chemical or optical properties of the electron acceptor is notrequired for the successful application of this invention.

These acceptor moieties are typically composed of one to 10, preferably1 to 4, fused cyclic hydrocarbon rings which may be substituted orunsubstituted. The cyclic hydrocarbons may independently have any ringsize ranging from 5- to 10-membered rings, but preferably include 5- or6-membered aromatic rings. The cyclic hydrocarbons may be fused in anyposition isomer.

Preferably, the acceptor or the donor moiety includes a group of generalformula IIIa or IIIb

wherein X, Y, Z, Q, V and W are independently selected from the atoms C,N, S, and O, X, Y, Z, Q, V and W are connected by either single ordouble bonds. R₁-R₆ are independently selected from the group of R₁-R₆are, independently from each other, selected from the group of —H, —O⁻,—OH, —OR′, —SH, —SR′, —NH₂, NO₂, —SO₃ ⁻, —SO₂ ⁻, —CN, —PO₃ ²⁻, —PO₂ ⁻,——COOH, —CO—R′, —COOR′, —CS—R′, CSO—R′, —COO⁻, —N═N—, halogen (—F, —Cl,—Br, —I), —NHR′, N(R′R″), or derivatives hereof, or hydrocarbyls orheterocyclyl as defined below. R′ and R″ is chosen from the possibledefinitions of R₁-R₆. At least one of X, Y, Z, Q, V and W can togetherwith the moiety R¹-R⁶ bound thereto also mean —CO—, —SO— or —SO₂—. Atleast one of R¹-R⁶ can also be a single or a double bond.

Hydrocarbyl comprises groups such as alkyl, alkenyl, alkynyl, eachhaving between 1 and 10 carbon (C)-atoms, aryl having 6-30 C-atoms, suchas phenyl, naphthyl, biphenyl, tolyl, anthracenyl, etc., andcombinations of these in different substitutions patterns. Thesehydrocarbyl groups may be straight chained or branched chained,symmetric or asymmetric, chiral or achiral, contain one or more heteroatoms selected among —N—, —NH—, —S—, —O—, and may also be fused. Thehydrocarbyl groups may be unsubstituted or substituted by one or more ofthe above mentioned R¹-R⁶.

Heterocyclyl are preferentially selected among cyclic aromatic ornon-aromatic moieties containing hetero atoms selected among —N—, —NH—,—S— and —O—, preferably selected from the group consisting of pyridyl,imidazolyl, pyrimidinyl, pyradazinolyl, quinolyl, acridinolyl, pyrrolyl,furyl, thienyl, isoxazolyl, oxazolyl and thiazolyl. The heterocyclylgroups may optionally be substituted by one or more of the abovementioned R¹-R⁶.

At least one of said moieties R¹-R⁶ is modified such that it is capableof binding to the backbone of the probe. Preferably the electronacceptor is bound via a free sigma bond at one of said moieties to saidbackbone. It is understood according to the present invention that theacceptor moiety is defined as the moiety which is capable andresponsible for accepting the electron. Any moiety between the backboneand the acceptor is defined to be a linker A.

While this is not the preferred case, the acceptor or/and donor moietycan include a complex of a transition metal chelated by one or moreligand moieties. Preferred transition metals are chosen from the groupiron, copper, ruthenium, rhenium and osmium.

More preferably, the electron acceptor or donor may contain a groupchosen from the class of imides. Imides may act as both acceptors anddonors. These compounds will contain the essential —(C═O)—N(R)—(C═O)—unit with the carbonyls placed in a hydrocarbon ring structure which isbonded to an aromatic ring system via conjugation of the carbonyls. Theimide is contained in a hydrocarbon ring which may be 5, 6, 7, 9membered but preferentially a 5 or 6 membered ring. The acceptor/donormay contain from 1-10 imides linked via the aromatic system. Anacceptor/donor may contain imides of the same ring size or containingimides of different ring sizes. The aromatic structure comprises of 1-10fused cyclic aromatic hydrocarbons and optionally substituted with thesubstituents (R). The cyclic hydrocarbons may be bonded/fused in anyposition isomer. The cyclic hydrocarbons may independently of each otherbe any ring size (5, 6, 10, 14 membered rings) but preferentially 5, 6membered aromatic rings which independently of each other may compriseone or several hetero atoms selected among —N—, —NH, —S— and —O—.

Preferred imides include groups of general formulae IVa-IVe.

wherein the definitions for R are selected from the possible definitionsof R¹-R⁶ of general formula IIIa and IIIb and wherein n and m is 0 or aninteger from 1 to 10 and k, r and l is 0 or an integer from 1 to 4. Itis clear that only so many substituents R are bound that the valency andcharge of the atom of attachment of the substituent R is not changed.

A further preferred moiety is chosen from the group ofn-alkyl-aza-aromatic compounds. N-alkyl-aza-aromatics may act as bothacceptors and donors (formula Va-d, vide infra). In these compounds thenitrogen atoms in the aromatic rings are alkylated. This formspositively charged molecules. The aromatic rings where the aza atoms arelocated can be 5, 6, 10, 14 membered rings, but preferentially 5 and 6membered rings. The acceptor/donor may contain 1 to 10 alkylated cyclicnitrogen atoms which are placed in an aromatic system of 1 to 10 fusedaromatic rings.

The fused cyclic hydrocarbon system may be composed of 5, 6, 10, 14membered aromatic rings, but preferentially 5 and 6 membered rings,which independently of each other may comprise one or several heteroatoms selected among —N—, —NH, —S— and —O—. The acceptor may be composedof alkylated cyclic nitrogen atoms of the same ring size and/ordifferent ring size. The aromatic hydrocarbons carrying the alkylatedcyclic nitrogen atoms and/or the fused aromatic ring not carrying thealkylated nitrogen atoms are preferentially substituted (R).

Within this group compounds of general formulae Va-Vd

wherein the definitions of the substituents of formula IV apply andwherein r is 0 or an integer of from 1 to 4.

Preferred as acceptors are compounds containing a quinoid structure.Such compounds are well-known to one skilled in the art. The quinonesespecially useful in this invention are composed of at least 2conjugated carbonyl groups (optionally thiono or azo) placed in the sameor in separate rings fused with 1-10 cyclic aromatic hydrocarbons andoptionally substituted with the substituents (R¹-R⁶) and having 2-20conjugated carbonyl groups in pairs, provided that the number ofconjugated carbonyl groups does not exceed twice the number of fusedcyclic hydrocarbons. The cyclic hydrocarbons may be fused in anyposition isomer. The cyclic hydrocarbons may independently of each otherbe any ring size but preferentially 5, 6 carbon atom membered aromaticrings which independently of each other may comprise one or severalhetero atoms selected among —N—, NH, —S— and —O—. The conjugatedcarbonyl groups may be located in any of these rings provided that thequinoid structure is maintained.

Especially preferred quinoid structures contain the general formulaeVIa-VIc.

wherein the definitions of the substituents are chosen from thedefinitions of formulae IV and wherein o and p are 0 or an integer from1 to 10 and t and q are 0 or an integer from 1 to 4. Preferred examplesof this group are anthraquinone and phenanthraquinone.

As electron donors, generally all compounds are useful which can in theground state or in the electronically excited state, or afterstimulation by some other means, be oxidized by an electron acceptor bygiving off an electron. Preferred donors are organic molecules, althoughorganic molecules containing metal ions are not excluded. Preferably,the donor molecules are chosen such that the electron deficiency causedby the oxidation has the consequence of causing a change to one or moreof its physical or chemical properties, whereby detection of theelectron deficiency (oxidation event) is possible. Typical donors willbe electron-rich fused aromatic systems carrying functional groups andwill optimally be substituted. The donor will preferably have anaromatic structure substituted by electron-donating groups. Therefore,the donors are preferably chosen from the general formulae III-V,wherein the substitution with electron-donating groups supersedes.

Electron-withdrawing groups or electron-attracting groups are preferablythe groups NO₂, —SO₃ ⁻, —SO₂ ⁻, —CN, —PO₃ ²⁻, —PO₂ ⁻, —COOH, —CO—R′,—COOR′, —CS—R′, CSO—R′, —COO⁻, halogen (—F, —Cl, —Br, —I), whileelectron-releasing groups/electron-donating groups are preferablyselected from the groups H, —O⁻, —OH, —OR′, —SH, —SR′, —NH₂, —N═N—, —I,—NHR′, N(R′R″).

Compounds preferentially acting as donors are compounds containing anaza-aromatic structure. Examples of these compounds are aromatic ringstructures containing nitrogen. The N-heterocyclic structure isbonded/fused with a cyclic hydrocarbon system composed of 5, 6, 10, 14membered aromatic rings, but preferentially 5 and 6 membered rings,which independently of each other may comprise one or several heteroatoms selected among —N—, —NH, —S— and —O—. The donor may be composed ofheterocyclic nitrogen containing rings of the same ring size and/ordifferent ring size. The aromatic hydrocarbons carrying the heterocyclicstructure and/or the bonded/fused aromatic ring system arepreferentially substituted.

Preferred aza-aromatic structures are compounds of general formulaeVIIa-VIIc

wherein the groups R, n, m, p, o, l, k, q, and r are chosen from thedefinition of formulae III to VI.

One possible donor moiety is the natural base guanine and derivativesthereof. It has been found in the present invention that guanine is aneffective donor, forming a chemically reactive species.

FIG. 1 shows schematically one embodiment of the present invention. Theprobe strand contains a donor moiety D having n electrons and anelectron acceptor A having m electrons covalently attached. Uponinduction of electron transfer (ET) one electron is transferred from thedonor moiety to the acceptor moiety, thereby changing the number ofelectrons in each moiety.

While applicants do not wish to be bound by this hypothesis, it appearsthat the use of nucleic acid analogues allows very selective control ofthe rate or efficiency of electron and hole transfer between a donor andan acceptor through modification of the distance or effectiveness ofelectron or hole transport through the π-electron stack of the DNA orthe nucleic acid analogue when bound to a complementary nucleic acidstrand. The orientation within such a complex is considered to be sorigid that the donor (acceptor) after excitation, or stimulation by somemeans, can donate (attract) an electron (hole) from a near-by acceptor(donor) or a nucleic acid base. The base where the electron or holecomes from is now oxidized or reduced and may bear a positive ornegative charge. The acceptor as a consequence is negatively charged.When a chemical moiety (here a nucleobase) is missing an electron, an“electron hole” (or just hole) is created in the molecule. The electronhole in the first base can then be filled by transfer of an electronfrom another base (see FIG. 5); this process is called hole-hopping. Byseveral such hole-hops, the excitation of the acceptor and subsequentelectron transfers yield oxidation of a distal position in the bindingproduct (for example, a hybrid between the nucleic acid analogue and thenucleic acid to be determined). Electron transfer may propagate in thestrand where it is initiated but may as well propagate in the oppositestrand. The rate or efficiency of electron or hole hopping will dependon its time scale and the time scale of chemical or physical processesthat compete for consumption of the excited or stimulated electron donoror acceptor.

FIG. 5 shows schematically the process of hole-hopping initiated byirradiation at 350 nm for a peptide nucleic acid (lower strand) havingan electron acceptor exemplified as Q within the sequence and replacinga base and a nucleic acid (upper strand) having bases B complementary tothe bases of the peptide nucleic acid. Electron transfer creates apositively charged base at the nucleic acid (B⁻ ⁺ ) by hole-hopping.This positive charge is preferentially transmitted to the 5′-terminalbase of a GG sequence, eventually causing chemical damage to this base.Upon treatment with piperidine, the linkage between the damaged base andthe nucleic acid backbone is cleaved. Release of the base then permitsscission of the analyte backbone, resulting in formation of smallerfragment molecules which can be readily detected by electrophoretic orchromatographic techniques. One aspect of the present invention is touse this electron transfer from an electron donor on the analyte to anelectron acceptor covalently linked to the probe molecule and subsequentcleavage of the analyte to detect hybridization. The present inventionis further based on the fact that this electron transfer is possibleonly if this complex between the nucleic acid to be determined and thenucleic acid analogue is formed, since single-stranded PNA probes reactwith analyte nucleic acids by electron transfer either not at all or ata much lower rate if the two strands are not hybridized. The rate orefficiency of electron transfer is significantly reduced for complexeshaving an internal mismatch. In this aspect of the current invention itis preferable that the electron transfer not be reversible.

The sample containing the nucleic acid to be determined is contactedwith the probe molecule in order to create the complex between thatprobe molecule and the nucleic acid to be determined. This complex willform via base-mediated hydrogen bonding. Peptide nucleic acids as probemolecules have the advantage that they can strand invade intodouble-stranded nucleic acids. Therefore, it is not even necessary toseparate double-stranded nucleic acids into single strands prior toadding the probe molecule. The binding of the probe molecule to thenucleic acid will be made according to conditions known to one skilledin the art. Such conditions are well described for peptide nucleicacids, for example in WO 92/20703, to which is made reference in thisrespect. Because in many cases, the amount of nucleic acids in thesample is not evident, it might be preferred to choose the probemolecule in the amount exceeding the highest expected amount of nucleicacids in the sample. However, in order to maintain a low level ofbackground signal, it may be advisable to choose the amount of probemolecules to be in the order of magnitude of the nucleic acid to beexpected. As described in WO 95/15971, there are several possibilitiesto determine the presence of any nucleic acid. The present invention,however, requires that at least one probe molecule is labeled by anelectron donor moiety or an electron acceptor moiety or both. The rulingprinciple is that within the complex containing the nucleic acid to bedetermined and the probe molecule, there is at least one electron donorand at least one electron acceptor moiety. While one of the electrondonor and electron acceptor moiety must be covalently bound to the probemolecule, at least one of the other species of electron transfermoieties can be located either within the same probe molecule, thenucleic acid to be determined or another probe molecule. This otherprobe molecule can either have a natural sugar phosphate backbone or anon-natural nucleic acid analogue backbone, while the nucleic acid to bedetermined preferably has the natural sugar phosphate backbone.

In a first embodiment, a complex is formed by a nucleic acid analogueprobe molecule having an electron acceptor moiety bound at an internalbackbone position, the nucleic acid to be determined or a nucleic acidbeing the product of an amplification process of a nucleic acid to bedetermined, and a further probe nucleic acid analogue probe moleculecontaining an electron donor moiety bound to the backbone at anon-terminal position, wherein the probe molecules are bound todifferent but adjacently located nucleotide sequences on the nucleicacid. It is important to avoid interrupting the base-stacking betweenthe binding sites of the two probes. As a general rule, the electrondonor and the electron acceptor of two different probe molecules shouldnot be at termini facing each other when bound to the nucleic acid. Itis obvious that groups at the termini facing each other would severelyhinder the capability of the probes to bind to adjacent sequences on thenucleic acid to be determined.

In a second embodiment, the nucleic acid is labeled to contain anelectron donor or electron acceptor moiety within the π stack. Asdescribed for the probe molecule, it is therefore preferred to insert aflat electron transfer moiety in place of a nucleobase. Such nucleicacids can for example be prepared by using a chemically synthesisedoligonucleotide primer having the electron transfer moiety near the3′-end. During amplification, if the original analyte nucleic acid ispresent, this primer is elongated by attachment of furthermononucleotides. The probe molecule is then chosen to bind to a positioncontaining both the part of the primer wherein the electron transfermoiety is located and a part of the adjacent extension product.

For the further determination of the complex, it is important that bythis binding process a complex is formed having at least one electronacceptor moiety and at least one electron donor moiety covalently boundto either of the probe molecules or the nucleic acid to be determined.Thus, the complex contains at least one electron acceptor and at leastone electron donor.

The formation of the complex is determined by inducing electron transferfrom an electron donor within that said complex to an electron acceptorwithin said complex and determining the occurrence of this electronictransfer as a measure of said nucleic acid. This induction of electronictransfer of course depends upon the choice of the electron donor or/andthe electron acceptor. There are at least two possibilities to startinduction. In the first case, the electron donor is activated to giveaway an electron to the π-stack within the complex. In the second case,the electron acceptor is activated to absorb an electron from a moietywithin the π-stack as described above. Generally, therefore the electronacceptor is changed in its electronic configuration, which will dependupon the π-systems involved. The activation process is described in thefollowing using the example of compounds chosen from the general formulaV. For example, anthraquinone derivatives have an nπ* excited stateelectronic configuration. Upon excitation by light, fast (≦10 ps)intersystem crossing to the triplet state will occur, precludingfluorescence emission. However, phosphorescence from the triplet statecan be observed at low temperatures. This emission is efficientlyquenched when the electron acceptor is intercalated into the π-stack ofthe complex formed due to rapid electron transfer from one of the basesadjacent to the intercalation site to the excited electron acceptor.When the electron acceptor is not located within the π-stack, electronicinteraction with the bases is weak at best and significantly reducedquenching occurs.

In a first embodiment therefore the occurrence of electronic transfer isdetermined by determining the degree of the reduction of quenchingcompared to the case when no nucleic acid to be determined is present.

The preferred mode of inducing electron transfer in this aspect of theinvention is therefore the inducement by irradiating the samplecontaining the complex with light at a wavelength wherein the activationof the electron acceptor is achieved. This will be for the electronacceptors mentioned above at wavelength of between 300 nm and 450 nm,most preferably between 330 nm and 360 nm. The vessel or containmentwherein the sample is maintained preferably is transparent to the lightused, such that the light yield is improved.

The generation of electron transfer (ET) is preferably done in anaqueous environment. Thus, the solvent used is preferentially water orwater containing up to 40% (v/v) of an organic medium, preferentially upto 10% (v/v) of an organic solvent such as methanol. Higher content oforganic solvents will not be used due to the instability of duplexesformed from nucleic acids and nucleic acid analogues, such as PNA, underthese conditions.

The solvent will preferentially be buffered and the pH range will be2-12. Buffers can be selected among the traditionally used buffers inbiological assays, but will often be phosphate buffers at pH 7. The saltconcentration in the buffer may vary but will preferentially be in therange of between 10 mM and 0.5 M.

It may be advantageous to mix the reaction mixture containing thecomplex during irradiation, such that homogeneous inducement isachieved.

Further possible ways for inducing electron transfer include stimulationof the electron acceptor at an electrode or by chemical means, or byirradiation with electrons, or by subjecting the samples to ionisingelectromagnetic radiation.

The time in which the stimulation conditions are maintained depends uponthe strength of stimulus. It is apparent to one skilled in the art thatif a strong stimulation source is used, inducement may be accomplishedin a shorter time than when using a low strength source. In order tokeep the time for the assay limited it is preferred to have irradiationtimes of less than 6 hours, more preferably less than 1 hour. Apractical lower limit for the stimulation time may be less than 1second. At high stimulation power there is a chance that damage toeither the sample or the sample's environment will occur.

The electron transfer induced is determined as a measure of the nucleicacid to be determined. The occurrence of the electron transfer can bemeasured by several methods. They may include determining the electrontransfer itself or may comprise the determination of any changesoccurring within the complex or its components caused by the electrontransfer. It is especially preferred to determine the changes within theelectron acceptor either immediately after the electron transfer or atsome time later when the process has reached its final state. The mostobvious change of the electron acceptor is the absorption of theelectron, changing the electronic configuration of the electron acceptoror donor. This change can be measured using either chemical orspectroscopic properties of the now electron-rich electron acceptor.Most preferred are determinations of the spectroscopic properties, suchas light absorption or emission at characteristic wavelengths (orquenching thereof). The electron-rich electron acceptor may also bedetermined by chemical or physical means such as transferring its excesselectron to a secondary acceptor or an electrode.

The electron donor has also changed its chemical and spectroscopiccharacteristics by losing an electron. For example, if guanine is theelectron donor, by losing an electron a reactive species is createdwhich renders the nucleic acid backbone susceptible to cleavagespecifically at the site of oxidation by piperidine treatment. Thischemical reaction is known to one skilled in the art. The occurrence ofelectronic transfer can then be determined by detecting nucleic acids orprobe molecules being shorter in length than expected. This can be madeeither by direct gel-electrophoresis or after sequencing as well as byliquid chromatography methods.

In case of electron donors changing their spectroscopic characteristicsupon losing an electron, the spectroscopic characteristics of themodified electron donor can be determined. Similar to the determinationof any changes in the acceptor, it is possible to spectroscopicallydetermine the presence of the created species using its light absorptionor fluorescence at characteristic wavelength.

A further indication of the electron transfer is quenching of a possibleluminescence of the electron donor or electron acceptor. It is wellknown to those skilled in the art that electronically excited states ofmany electron donors or electron acceptors emit light. Electron transferreactions can effectively quench this emission. In one aspect of thisinvention, quenching of the luminescence of the excited electron donoror the excited electron acceptor is the means by which formation of thecomplex between the probe and the nucleic acid is determined. Forexample, luminescence from the electron donor or electron acceptor willbe reduced by formation of the complex if the rate or efficiency ofelectron transfer is increased by complex formation. In the preferredapplication of this invention, the luminescence from the excited donoror acceptor is increased by complex formation because the rate orefficiency of electron transfer has decreased as a consequence ofcomplex formation.

In addition to the dependence of the reaction rate on the free energy,reaction rate will also be dependent on distance. In particular, thereaction rate is expected to decrease exponentially with distance:

k_(et∞)e^(−βr)

where r is the distance separating the donor and acceptor and β is adampening factor with units of distance ⁻¹. The dampening factor β′ is ameasure of the conductivity of the medium: a low β will allow electrontransfer to occur over very long distances while a high β will permitelectron transfer at short distances only.

There is currently some controversy regarding the β value for duplexDNA: Barton and coworkers have suggested that β could be very low (<0.2Å⁻¹) (Murphy, C. J.; Arkin, M. R.; Jenkins, Y.; Ghatlia, N. D.;Bossmann, S. H.; Turro, N. J.; Barton, J. K.; Science 1995, 262,1025-1029), while Harriman and Brun have measured a β value of 0.88 Å⁻¹(Brun, A. M.; Harriman, A. J. Am. Chem. Soc. 1992, 114, 3656-3660). TheBarton value would suggest that duplex DNA is an exceptional conductorwhile the Harriman/Brun value would suggest that duplex DNA is anordinary conductor. The Barton value is not well-substantiated byexperiment (i.e. the occurrence of electron transfer has not beendemonstrated) so the Harriman/Brun value seems more reliable at thistime. It should also be noted that the β values for PNA-DNA and DNA-DNAcould be quite different since the electron which is transferred ispassing through the π-stack and the structures of the π-stacks are quitedifferent for the two types of duplexes.

If one assumes a “normal” β value of ca. 1.0 Å⁻¹, then a change indistance from 10 to 20 Å would decrease the electron transfer rate bymore than four orders of magnitude. Such a strong dependence of theelectron transfer rate on distance is the foundation of our distinctionbetween bioconductors and bioinsulators, particularly with respect tothe severe time constraints imposed on the system by usingphotoinduction. The excited state species used as either the donor oracceptor will exist for a relatively short time. In a preferredembodiment, the donor absorbs a photon and has a relatively shortlifetime, meaning it will relax to the ground state within a fewnanoseconds of excitation. This relaxation can occur by severalmechanisms but the most important pathway is fluorescence, in whichphoton of light is emitted. In the presence of a suitable electronacceptor, resulting in a decrease in the fluorescence. “Suitability” isdetermined by the acceptor reduction potential: ideally, the potentialwill be sufficiently low to yield a negative free energy for electrontransfer. If the potential is too high, then the electron transfer willbe too slow to compete effectively with fluorescence and littlequenching will result. Therefore, the strong dependence of electrontransfer on distance indicate that, with the proper donor/acceptor pair,one can convert a system from a bioconductor to a bioinsulator simply byincreasing the donor-acceptor separation distance. The key is toincrease the distance (decrease the reaction rate) until the electrontransfer becomes too slow to compete with fluorescence.

In the PNA hairpin system we take advantage of the large increase indistance separating the donor and acceptor after hybridization in orderto inhibit electron transfer. The fact that we observed significantlymore fluorescence after hybridization than in the hairpin conformationindicates that β is not very small. The ideal fluorescer will have ashort lifetime (the shorter the better), a high quantum yield, a lowintersystem crossing rate, energetics so that energy transfer is fast,be flat so that it intercalates in the hairpin and the duplex withoutmessing up base-pair recognition by hydrogen bonding, absorb where theanthraquinone does not, fluoresce in the “red” region of the spectrum,and it should also be easy to synthesize.

Such ideal fluorescor would give a substantially black background whenno nucleic acid to be determined is present and bright fluorescence inthe complex of probe and nucleic acid.

In a preferred mode of the method of the present invention, the rate orefficiency of the electron transfer is different in the probe and in theprobe when bound to the nucleic acid. In this case the electron donorand acceptor are preferably bound covalently to the same probe molecule,most preferred at a defined, fixed first distance. Upon binding of theprobe to the nucleic acid to be determined, the distance from theelectron donor to the electron acceptor is altered, preferablyincreased, thus decreasing the reaction rate of the electron transfer.The increased distance results in a decreased rate of electron transferand a concomminant increase in detectable signal. The detectable processwill then serve as an indication or measure of the electron transfer andthus of the presence or absence of a nucleic acid to be detected.

A preferred method of the invention therefore is a method wherein thedistance between acceptor and donor is a probe is altered upon bindingto the nucleic acid, preferably by more than 5 base units in a stack.

In order to evaluate the results from determining of the occurrence ofthe electronic transfer, one skilled in the art will correlate thesignals or results received from experiments wherein the amount orpresence of a nucleic acid to be determined is defined. This can then bemade by usual calibration experiments, for example, using standardsamples containing different specified amounts of nucleic acids to bedetermined in subjecting the standards to the same conditions as thesample suspected to contain the nucleic acid to be determined.Qualitative and quantitative determination is possible.

An important feature of the formation of the probe-analyte complex isthat the distance between the donor and acceptor is relatively fixed.

While these are the core steps of the method of the present invention,this method can be adapted to known convenient formats, for example, byintroducing this concept into assays wherein the nucleic acid to bedetermined is determined in an immobilized state. A very advantageousformat of the present invention is disclosed in FIG. 2. In thishomogeneous (i.e. single solution) format, the sample containing thenucleic acid to be determined (comp. target DNA) and nucleic acids notto be determined (non-comp. target) in soluble form, the probe moleculecontaining the electron acceptor and an electron donor are mixed. Onlynucleic acids to be determined will hybridize to the specific probemolecule. Nucleic acids not complementary to the probe molecule will nothybridize. Therefore, after inducement of electron transfer, there willbe higher or lower light emission from the donor or acceptor if thenucleic acid to be determined was present.

In FIG. 3, the same homogeneous format is described wherein one probecontaining an acceptor and another probe containing a donor is used.Only binding of both probes adjacent to each other, whereby the electrontransfer is capable of propagating from donor to acceptor will yield achange in light emission.

In FIG. 4, a heterogeneous assay format is disclosed. The probe moleculecontaining both an electron acceptor (acc.) and an electron donor(Donor) is bound to a solid phase, for example, a polystyrene surface.This can be accomplished either by covalent bonding or usingstreptavidin-coated surfaces and biotin-labeled probes. Upon excitationof the electron acceptor, electron transfer is induced on the solidphase, and can be detected by identifying the modified donor only if thenucleic acid to be determined was bound to the solid phase nearbase-pairing via the immobilized probe molecule. The assay format usingan immobilized probe is especially advantageous if the sample containsfurther ingredients disturbing the irradiation of a detection, forexample, by absorbing light in the range of the irradiation or emissionwavelength.

In an embodiment the nucleic acid to be determined is bound by nucleicacid analyte probe molecules capable of forming a hairpin structure. Inthis design, the electron donor (Do) and acceptor (Ac) are initiallybrought into relatively close proximity by the nature of the hairpinstructure (FIG. 6). The close location of the electron transfer moietieswill generally lead to high electron transfer rates and efficienciesbetween these two groups. Thus, spectroscopic changes induced byelectron transfer will be pronounced. This could, for instance, beeither luminescence initiation or luminescence quenching by electrontransfer. A preferred application of this invention is the case whenduplex DNA or the DNA-PNA complex of the probe and the nucleic acid tobe determined has the characteristic of a bioinsulator. In this casewhen the hairpin probe is hybridised to a target nucleic acid (e.g. aPCR amplicon) electron transfer rates will be reduced compared with therate in the probe itself and, therefore, less or no luminescencequenching will be observed from the complex than from the probe.

A hairpin structure is synthesized preferentially in a parallel mode. Itconsists of two hybridisable segments and a hinge segment (a segmentlinking the bybridisable segments). The two hybridisable segments arecomplementary to each other and to two non-overlapping segments of thenucleic acid to be determined. Due to the parallel synthesis of thewhole hairpin, the two segments will form an antiparallel duplex. Thetwo hybridisable segments are preferentially composed of 6-12 monomerseach, each segment containing an electron transfer moiety capable or notcapable of forming hydrogen bonds. The electron transfer moieties arepreferentially placed in such a way that they are juxtaposed in thehairpin structure on that one of them is an electron donor and one ofthem is an electron acceptor. The close positioning of the electrontransfer moieties makes electron transfer as optimal as possible. Thepreferred distance of the electron transfer moieties on differentsegments is between 1 and 10, more preferably 1 and 5 when the segmentsare hybridized to each other (a distance of 0 meaning base positionsforming hydrogen bonds with each other, if bases were present at thisposition). The hinge segment is composed of 2-7 monomers, preferentiallycomposed of 3-5 monomers.

Stem-loop (hairpin) RNA and DNA structures are known to form readilyfrom appropriate nucleic acid sequences. We have now shown that PNA alsoform hairpin structures. Characteristically, the thermodynamic stabilityof the PNA structure, revealed by its melting temperature, is greaterthan that observed for DNA which contains an identical sequence. Inparticular, for cases where a change in spatial relationship providesthe detected signal, PNA will provide greater spatial differentiationwith fewer base pairs than the comparable DNA-based sequence.Exploitation of this advantage requires the ability to prepare specificPNA sequences covalently linked to “reporter” units that do notcompletely disrupt the hairpin structure. We have developed synthetictechnology to incorporate reporter groups linked to internal PNAposition.

The hairpin PNA contains an electron donor, for example an amnoacridinegroup in place of one of the bases on the PNA backbone. By design, theacridine group falls within the duplex region of the PNA hairpin. Themelting behavior and, especially, circular dichroism spectroscopyindicate that incorporation of the acridine does not prohibit hairpinformation. Similarly, the evidence indicates that a PNA, which containstwo non-nucleic acid groups (an acridine and an anthraquinone) bound inthe duplex region of the hairpin, also maintains the ability to formstem-loop structures.

One of the key requirements to be satisfied by a nucleic acid biosensoris selectivity. The probe must recognize its complement selectively andinitiate a unique, sensible event. The unrivaled ability of PNA to formduplex, and higher order, structures with complementary DNA and RNAsuggested that their modification to include reporter groups would notdestroy the recognition ability. The experiments described below revealthis to be the case. The melting behavior observed for mixtures of PNAand, its reaction with a dodecamer DNA complementary to only the loopand a portion of the stem region of the PNA. The melting behavior andthe CD spectroscopy of the PNA/DNA structure indicates formation of ahybrid structure that has opened the PNA hairpin.

The preferred aminoacridine unit of non-nucleotide substituent ispreferably linked to the PNA backbone through a carbon-nitrogen bond toits 9-amino group. While the properties of 9-aminoacridine has beenextensively studied, there are only a few reports concerning the monoN-alkylated aminoacridines. We use 9-(N-methylamino)acridine as a modelfor the acridine group of Ar. In acetonitrile solution, the fluorescencequantum yield (Φ_(fl)) for 9-aminoacridine is 0.96 and its singletlifetime is 15.8 ns (Kubota et al., J. Phys. Chem. 84, 2855-2861(1980)). In contrast, under these conditions the (Φ_(fl)) for9-(N-methylamino)acridine is only 0.011 and its singlet lifetime iscorrespondingly reduced to 2 ns. This dramatic change in photophysicalproperties is mirrored in a comparison of photoelectron spectra (Li etal., Zh. Obshch. Khim 61, 186-191, 1991). These effects are attributedto a change in excited state character caused by loss of overlap of theacridine π-electron system with the non-bonding electrons of the aminonitrogen atom due to steric effects when the methyl group of9-(N-methylamino) derivative is co-planar with the acridine. The reducedlifetime of the methylaminoacridine makes it less susceptible tofluorescence quenching by guanine and adenine (Kubota, 1980).Consequently, we observe little difference in the aminoacridinefluorescence intensity between the hairpin and linear forms of PNA.

The aminoacridine fluorescence of the PNA is strongly quenched when itis in its hairpin form. We attribute this to electron transfer from theexcited singlet state of the acridine to the nearby anthraquinone group.The oxidation potential of 9-(N-methylamino)acridine is reported to be0.21 V vs SCE (Shen et al., Science in Clinc. (Ser. B) 35, 137-145(1992)). The reduction potential of anthraquinone derivatives closelyrelated to Q is −0.58 V vs SCE (Breslin et al., J. Am. Chem. Soc. 118,2311-2319 (1996)). The energy of the singlet excited state of theaminoacridine in PNA calculated from its fluorescence spectrum is 3.45eV. Application of the Weller equation (Rehm et al., Isr. J. Chem. 8,259 (1970)) ignoring the Coulomb work term, which should be negligible,reveals that the free energy for electron transfer (ΔG_(et)) is −2.66 eV for transfer of an electron from the singlet excited state of theaminoacridine to the anthraquinone. This reaction is sufficientlyexothermic for it to occur at every encounter between the excitedacridine and the quinone.

There is weak residual aminoacridine fluorescence from the hairpin formof the PNA. We attribute this to multiple conformations of the quinoneand acridine groups as is suggested by its multi-phase melting behavior.If the excited state aminoacridine group is in contact with the quinone,then quenching should be nearly instantaneous and complete. The precisemeaning of “contact” is not clearly defined by these experiments. Wetake it, provisionally, to mean that both the quinone and acridine areintercalated within the duplex region of the PNA hairpin. In thisregard, if either the quinone or acridine is extrahelical when theacridine is excited, it seems unlikely that binding within the duplexwill occur within the ca. 2 ns lifetime of the excited state. The datashow that the aminoacridine fluorescence increase ca. 3.3 fold when thePNA is converted from its hairpin to linear form. Interpreted within themulti-conformational model, his finding indicates the ratio of PNAstructures where there is quinone/acridine are in contact to those whereone group is extrahelical is ca. 3.3:1.

The data show that the reaction of the hairpin PNA with either DNA orDNA results in a ca. 6.5 fold increase in aminoacridine fluorescenceintensity. We attribute this to conversion of the hairpin form of thePNA to a linear hybrid duplex in which the acridine and quinone are notlonger in contact. There has been considerable recent discussionconcerning the ability of DNA to conduct holes (radical cations) andelectrons (Beratan et al., Chemistry and Biology 4, 3-8 (1997)). Bartonand co-workers (Arkin et al., Chem. & Biology 4, 389-400 (1997);Dandliker et al., Science 275, 1465-1468 (1997), Hall et al., Nature382, 731-735 (1996)) have argued for very rapid transport. Meade andco-workers (Meade et al., Angew. Chem. Int. Ed. Engl. 34, 352-354(1995)) report a slower rate. Our findings suggest that on the shorttime scale of the aminoacridine lifetime, the PNA/DNA hybrid duplexbehaves as an insulator not a conductor. Thus, whether intercalated ornot, the quinone does quench the aminoacridine fluorescence when the twogroups are separated by twelve pairs in the hybrid duplex.

The use of PNA in the construction of probes to sense nucleic acidsoffers several advantages over using DNA as the sequence-selectiverecognition element. PNA/DNA hybrid duplexes provide exquisitesingle-base-mismatch selectivity (Wang et al., Anal. Chem. Acta 344,111-118 (1997)) and offer greater stability than their DNA/DNA orDNA/RNA counterparts. This is especially advantageous in the hairpinformat of the molecular beacons. The duplex region of the hairpin willgenerally not be fully complementary to the targeted nucleic acid, andthis region can be shorter for a PNA probe than for a DNA probe. Also,since PNA is unnatural, there are no nuclease enzymes that destroy it.This offers a special advantage in homogeneous assays because fewersample-preparation steps will be required. Finally, and perhaps ofgreatest significance, the fluorescer and quencher groups can be easilyplaced in internal positions in PNA. PNA is easily synthesized bystandard peptide methods and the required monomeric derivatives arereadily prepared by simple reactions. Internal positions for thefluorescer and quencher provide the prospect of high sensitivity sincethere will be no “fraying” that is likely to occur when the fluorescerand quencher are bound at opposite termini of the hairpin duplex.

In another embodiment the close electronic interaction between the donorand the acceptor is also used (see FIG. 7). In this embodiment theanalyte specific probe (PNA2) contains the donor (or the acceptor). Asecond probe (PNA1) containing the other electron transfer moiety iscomplementary to a part of the first probe. When these are hybridisedthe interaction between the acceptor and the donor is as outlined above.When the analyte is introduced to the mixture, the short acceptor probewill be displaced by the analyte thus preventing the electron transferinteraction between the donor and the acceptor. Depending on the choiceof donor/acceptor this could lead to either luminescence initiation orquenching.

Further subject matter of the invention are molecules of the generalformulae I and II.

The compounds of the general formula I can be used advantageously in theabove-disclosed method for determining a nucleic acid in a sample as thenucleic acid analogue probe molecule. These compounds can be preparedanalogous to the synthesis described in WO 92/20702, WO 94/25477, WO96/20212, EP-0 672 677 and EP-0 700 928, just replacing one or moremonomers used in the oligomerisation with one ore more monomerscontaining non-nucleobase containing electron acceptors ornon-nucleobase containing electron donors. A preferred way to producethe compounds of general formula I is the step-wise chemical synthesisaccording to WO 92/20702, incorporating as a monomer a compound of thegeneral formula VIIIa-VIIIc

wherein the definitions of A, B, C and D are chosen from the definitionsof A¹-A^(n), B¹-B^(n), C¹-C^(n) and D¹-D^(n) in formula I, respectivelywith the condition that any amino groups therein may be protected byamino protecting groups; E is COOH CSOH, SOOH, SO₂OH or an activatedderivative thereof; F is NHR³ or NPgaR³, where R³ is as defined aboveand Pga is an amino protecting group and L is a non-nucleobase electrondonor or acceptor moiety.

Preferred monomers are amino acids having formula (IX)

wherein X′ is a carboxylic acid protection group or hydrogen, R^(7′) isindependently selected from the group consisting of hydrogen and theside chains of naturally occurring alpha amino acids, or amino-protectedand/or acid terminal activated derivatives thereof.

Therefore, a preferred method of preparing a molecule of general formulaI is characterized in that a molecule of general formula VIIIa-VIIIc orIX wherein X′ is hydrogen is reacted with a compound of general formulaX

H₂N—K  (X)

wherein

K is a solid phase, like amino modified glass, or a solid phase bound orfree compound of general formula I or II or a compound of formula I inprotected form. These compounds can further be modified by attachingmonomeric units not containing an electron transfer moiety.

Any reactive groups like primary amino groups or hydroxyl groups arepreferably protected by removable or non-removable protecting groups,like benzyloxycarbonyl, Boc or Fmoc if they are not intended to react inthe elongation reaction.

The following examples shall explain the invention in more detail usingPNA as an example of a probe molecule. The PNA oligomers described aboveare synthesised by solid phase synthesis procedures. The preferred solidsupport is polystyrene but also other solid supports such as ®Tentageland ®Controlled Pore Glass may be used. The synthesis proceeds from theC-terminal position of the PNA and proceeds via couplings of monomersand/or amino acids protected with acid labile protecting groups.Reactive groups on the bases/side chains are also protected. Afterdeprotection of the moiety directly linked to the solid support thesubsequent building blocks (monomers/amino acids) are then coupled inthe desired sequence. The deprotection of the N-terminal position isperformed prior to coupling of the following monomeric unit.

The coupling of the monomeric unit is performed via activation of thecarboxylic residue. Such activation is well known to those skilled inthis art and, as examples, are: HATU, HBTU, and carbodiimides. Thecoupling is performed in solvents normally used in peptide chemistry,that is NMP, DMF, acetonitrile, pyridine, dichloromethane or mixturesthereof Activation enhancers such as DMAP may be added. The activatedmonomeric derivative will be used in excess to the previouslydeprotected residue and typically in a 2 to 10 times excess. If a highcoupling yield is detected (typically >99%) the coupling step will befollowed by a capping step, which comprises treatment of the coupledresin bound oligomer with acetic anhydride. The capping mixturecomprises 2-20% (v/v) acetic anhydride in NMP/pyridine. Ifunsatisfactory coupling is detected the coupling step will be repeatedto obtain a high coupling yield.

Coupling of a conjugate containing a carboxylic acid can be doneanalogously to the above described procedure. Other ligands notcontaining a carboxylic acid can also be linked to the resin boundoligomers. Such other linkages can be performed via isocyanates,isothiocyanates (e.g. fluorescein isothiocyanate), carbonic acid activeesters, and sulphonyl chlorides.

The described stepwise synthesis of PNA can be performed manually orautomatically. In the latter case commercially available synthesisers(e.g. peptide synthesisers, multiple peptide synthesisers, DNAsynthesisers) can be used, provided that the hardware in the instrumentsis compatible with the chemistry used during the PNA synthesis.

The synthesised oligomer is finally removed from the resin. This ispreferentially done by treatment with strong acid, such as TFMSA or HF.The purification of the liberated oligomer is done by HPLC and/or ionexchange. The identity of the pure material is confirmed by massspectrometry and HPLC.

The letter designation of amino acids and the orientation of PNA followsthe traditionally used nomenclature.

EXAMPLES

Abbreviations Q1 Designates a monomeric unit composed by:3,6-Diaza-(N³-2- anthraquinoyl)-N⁶-boc-hexanoic acid Q2 Designates amonomeric unit composed by: 3,6-Diaza(N³-boc-aminoethyl)-4,7-dioxo-7-(2-anthraquinyl)-heptanoic acid Boctert-Butyloxycarbonyl R1 Designates a monomeric unit composed by:3,7-diaza-(N³-boc- aminoethyl)-(N⁷-9-acridinyl)-4-oxo-heptanoic acid R2Designates a monomeric unit composed by: 3,10-diaza-(N³-boc-aminoethyl)-N¹⁰-9-acridinyl)-4-oxo-decanoic acid DCCDicyclohexylcarbodiimide DCM Dichloromethane DIEA DiisopropylethylamineDMAP Dimethylaminopyridine DMF Dimethylformamide ET Electron TransferHATU O-(7-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate HBTUO-(7-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium- hexafluorophosphateHOBT 1-Hydroxybenzotriazole NMP N-methylpyrrolidinone NMR Nuclearmagnetic resonance PNA Peptide nucleic acid according to WO 92/20702 TFATrifluoroacetic acid TFMSA Trifluoromethanesulphonic acid HF Hydrogenfluoride AQ Anthraquinones (genus)

Oligomers are designated according to the PNA numbers assigned by PNADiagnostics.

Example 1 Synthesis of 3,6-Diaza-(N³-2-anthraquinoyl)-N⁶-Boc-hexanoicacid (Q₁, FIG. 8)

Anthraquinone-2-carboxylic acid (2 g, 7.9 mmol), DCC (1.7 g, 8.3 mmol),HOBT (1.08 g, 8.0 mmol) and methyl (N-(2-Boc-aminoethyl))glycinate (2 g,8.6 mmol) were dissolved in DMF (25 mL) and stirred at room temperatureovernight. The reaction mixture was filtered and the filtrate was washedwith DCM (2×25 mL). The solution was extracted with diluted NaHCO₃ (3×25mL), 2 M NaHSO₄ (2×25 mL) and brine. The organic phase was dried withMgSO₄, filtered and evaporated to dryness under reduced pressure. Theyellow foam was dissolved in THF (10 mL) and 1 M LiOH (30 mL) was added.The mixture was stirred for 2 h. THF was removed from the solution underreduced pressure and pH was adjusted to 2.8 with 2 M NaHSO₄. Theprecipitate was extracted with DCM (2×25 mL), evaporated to dryness andthen redissolved in ethyl acetate (3 mL). The ethyl acetate solution waspoured into hexane (150 mL) whereby the target molecule precipitated.Yield 3.1 g (87%).

H¹-NMR DMSO-d₆ δ: 7.81-8.30 (m, 7 H, aromatics); 6.98 and 7.72 (m, 1 H,BocNH); 4.17 and 3.97 (s, 2 H, CH₂O); 3.52 and 3.24 (m, 2 H, CH2); 3.21and 3.02 (m, 2 H, CH₂) 1.17 and 1.19 (s, 9 H, Boc).

Example 2 Synthesis of3,6-Diaza(N³-Boc-aminoethyl)-4,7-dioxo-7-(2-anthraquinyl)-heptanoic acid(Q₂, FIG. 9)

Methyl 4-(2-anthraquinyl)-4-oxo-3-aza-butanoate

Anthraquinone-2-carboxylic acid (3 g, 11.9 mmol), DIEA (3.1 ml, 24 mmol)and HATU (4.5 g, 11.9 mmol) in DCM (30 mL) was stirred for 10 min atroom temperature. Methyl glycinate hydrochloride (1.5 g, 11.9 mmol) wasadded after which the product started to precipitate. The mixture wasstirred for an additional 3 h. The precipitated material was collectedby filtration. The volume of the mother liquor was reduced to ⅓ and anadditional crop was collected. Yield 3.65 g (93%).

4-(2-Anthraquinyl)-4-oxo-3-aza-butanoic acid

Methyl (2-anthraquinoyl)-3-aza-butanoate (3.65 g, 11.3 mmol) was stirredfor 1 h at room temperature in 1 M LiOH (20 mL) and THF (3 mL) wherebythe starting material dissolved. pH of the solution was adjusted to 2.8with 2 M NaHSO₄ and the target molecule precipitated. The organicsolvents were removed from the suspension under reduced pressure and theprecipitate was collected by filtration and dried. Yield 3.5 g (100%).

H¹-NMR DMSO-d₆ δ: 9.37 (t, 1 H, NH); 8.67 (d, 1 H, H-laq); 8.36 (dd, 1H, H-3aq); 8.30 and 8.28 (1 H, H-4aq); 8.23 (m, 2 H, H-5 and H-8aq);7.95 (m, 2 H, H-6 and H-7aq); 4.00 (d, 2 H, CH₂).

3,6-Diaza(N³-Boc-aminoethyl)-4,7-dioxo-7-(2-anthraquinyl)-heptanoic acid(Q₂)

4-(2-Anthraquinoyl)-4-oxo-3-aza-butanoic acid (3.5 g 11.5 mmol), DCC(2.6 g, 12.65 mmol), HOBT (1.7 g, 12.65 mmol) and methyl(N-(2-Boc-aminoethyl))glycinate (3.2 g, 13.8 mmol) was stirred in DMF(50 mL) at room temperature for 48 h. The reaction mixture was filteredand the residue was washed with DCM (2×30 mL). The organic phase wasextracted with diluted NaHCO₃ (3×30 mL), 2 M NaHSO₄ (2×30 mL), brine,dried with MgSO₄ and evaporated to dryness. Crystallization from ethylacetate gave 4.0 g (67%) of methyl3,6-Diaza(N³-Boc-aminoethyl)-4,7-dioxo-7-(2-anthraquinyl)-heptanoate.The methyl3-aza-3-(N-2-Boc-aminoethyl)-4,7-dioxo-6-aza-7-(2-anthraquinoyl)-heptanoatewas hydrolysed at room temperature in 1 M LiOH and 10% THF. The pH ofthe solution was adjusted to 2.8 with 2 M NaHSO₄ and the target moleculeprecipitated as an oil. The oil was extracted with ethyl acetate and theorganic phase was dried with MgSO₄. The volume was reduced to 2 mL andhexane (250 mL) was added with stirring, whereby3-aza-3-(N-2-Boc-aminoethyl)-4,7-dioxo-6-aza-7-(2-anthraquinoyl)-heptanoicacid precipitated. Yield 3.7 g (92%).

H¹-NMR DMSO-d₆ δ: 12.64 (s, 1 H, COOH); 9.14 (m, 1 H, NH); 8.69 (d, 1 H,H-laq); 8.67 and 8.35 (dd, 1 H, H-3aq); 8.32 and 8.30 (dd, 1 H, H-4aq);8.25 (m, 2 H, H-5 and H-8aq); 7.69 (m, 2†H, H-6 and H-7aq); 6.90 and6.75 (t, 1 H, BocNH); 4.24 (d, 2 H, CH₂CO); 4.24 and 4.12 (dd, 2 H,CH₂CO); 3.90 (s, 2 H, CH₂CO); 3.44, 3.17 and 3.06 (m, 2 H, CH₂); 1.40and 1.38 (s, 9 H, Boc).

Example 3 Synthesis of R₁ (FIG. 10)

9-(2-Carboxyethyl)aminoacridine

β-Alanine (1 g, 11.2 mmol) was added to a solution of 9-phenoxyacridine(2.7 g, 10 mmol) in phenol (15 g). The suspension was stirred at 120° C.for 2 h. The solution was cooled to room temperature and poured intoether whereby the product precipitated as a yellow-green solid. It wastriturated with hot ethanol, filtered and then washed with ethanol,giving crude 9-(2-carboxyethyl)aminoacridine. Yield 1.9 g (71%).

H¹-NMR DMSO-d₆ δ: 8.27 (d, 2 H, acr. 1,8); 7.59 (m, 4 H, acr. 2,3,6,7);7.26 (m, 2 H, acr. 4, 5); 4.05 (t, 2 H, CH₂CO); 2.82 (m, 2 H, CH₂N).C₁₆H₁₄N₂O₂: calc/found: 266/266.

3,7-diaza-(N³-Boc-aminoethyl)-(N⁷-9-acridinyl)-4-oxo-heptanoic acid (R₁)

A mixture of 9-(2-carboxyethyl)aminoacridine (1.7 g 6.4 mmol), DIEA (1.8g 13.8 mmol) and HATU 2.68 g, 7 mmol) was stirred in DMF (40 ml) at roomtemperature for 10 min. Methyl-Boc-aminoethylglycinate (1.8 g, 7.8 mmol)was added and the solution was stirred at 60° C. for 4 h. The solutionwas cooled and DCM (100 ml) was added. Subsequently the solution wasextracted with 0.5 M NaHCO₃ (3×40 ml), 2 M NaHSO₄ (2×40 ml) and brine.The organic phase was dried with MgSO₄ and evaporated to dryness. Theresidue was dissolved in THF/1 M LiOH (2/10) 60 ml The pH of thesolution was adjusted to 2.8 with NaHSO₄ whereby an oil precipitated.The water phase was decanted off and the oil was dissolved in ethanol(10 ml). The solution was purred into ether and3,7-diaza-(N³-Boc-aminoethyl)-(N⁷-9-acridinyl)-4-oxo-heptanoic acid (R₁)precipitated. Yield 2.3 g (77%).

H¹-NMR DMSO-d6 δ: C₁₆H₃₀N₄O₅: 8.50 (dd, 2 H, acr. 1, 8); 7.91 (m, 2 H,acr. 2, 7); 7.84 (m, 3 H, acr. 3,6 and 9 NH); 7.49 (m, 2 H, acr. 3,6);6.89 and 6.76 (m, 1 H, BocNH); 4.28 (m, 2 H, CH₂CO); 4.05 and 3.92 (s, 2H, CH₂CO); 2.82 (m, 2 H, CH₂N). C₂₅H₃₀N₄O₅: calc/found: 466/466

Example 4 Synthesis of3,10-diaza-(N³-Boc-aminoethyl)-(N¹⁰-9-acridinyl)-4-oxo-decanoic acid(R₂, FIG. 11)

9-(5-Carboxypentyl)-aminoacridine

6-Aminohexanoic acid (1.47 g, 11.2 mmol) was added to a solution of9-phenoxyacridine (2.7 g, 10 mmol) in phenol (15 g). The suspension wasstirred at 120° C. for 2 h. The solution was cooled to room temperatureand poured into ether whereby the product precipitated as a yellow-greensolid. It was triturated with hot ethanol, filtered then washed withethanol, giving crude 9-(5-carboxypentyl) Aminoacridine. Yield 2.4 g(78%).

H¹-NMR DMSO-d₆ δ: 8.27 (d, 2 H, acr. 1, 8); 7.61 (m, 4 H, acr. 2,3,6,7);7.26 (m, 2 H acr. 4, 5); 3.79 (t, 2 H, CH₂CO); 2.16 (t, 2 H, CH₂N);1.72, 1.50 and 1.37 (m, 2 H, CH₂). C₁₉H₂₀N₂O₂: calc/found: 308/308.

3,10-diaza-(N³-Boc-aminoethyl)-(N¹⁰-9-acridinyl)-4-oxo-decanoic acid(R₂)

A mixture of 9-(5-carboxypentyl)aminoacridine (1 g, 3.2 mmol), DIEA (0.9g, 6.9 mmol) and HATU (1.34 g, 3.5 mmol) was stirred at room temperaturefor 10 min in DMF (30 mL). Methyl-Boc-aminoethylglycinate (0.9 g, 3.8mmol) was added and the solution was stirred at 60° C. for 4†h. Thesolution was cooled and DCM (60 mL) was added. Subsequently the solutionwas extracted with 0.5 M NaHCO₃ (3×30 mL), 2 M NaHSO₄ (2×30 mL) andbrine. The organic phase was dried with MgSO₄ and evaporated to dryness.The residue was dissolved in THF/1M LiOH (2/10) (40 mL). The pH of thesolution was adjusted to 2.8 with NaHSO₄ whereby an oil precipitated.The water phase was decanted off and the oil was dissolved in ethanol (5mL). The solution was then poured into ether and3N-(Boc-arninoethyl)-10N-(9-acridinyl)-3,10-diaza-4-oxy-nonanicacidprecipitated. Yield 1.2 g (76%).

H¹-NMR DMSO-d₆ δ: 8.51 (d, 2 H, acr. 1, 8); 7.90 (m, 2 H, acr. 2, 7);7.82 (m, 2 H, acr. 3, 6); 7.71 (m, 1 H, NH); 6.83 and 6.45 (m, 1 H,BocNH); 7.51 (m, 2 H, acr. 4, 5); 4.01 (m, 2 H, CH₂CO); 3.98 and 3.88(s, 2 H, CH₂CO); 3.23, 3.00, 2.30, 2.03 and 1.88 (m, 2 H, CH₂); 2.16 (t,2†H, CH₂N); C₂₇H₃₆N₄O₅: calc/found: 508/508.

Example 5 Synthesis of 3,6-Diaza-(N³-acetyl, N⁶-Boc)-hexanoic acid

To a solution of methyl N-(Boc-aminoethyl)glycinate (2 g, 8.6 mmol) inDCM (30 mL) was added acetic anhydride (0.9 g, 8.8 mmol) and pyridine (1mL). The mixture was stirred at room temperature for 2 h. The reactionwas extracted with 0.5 M NaHCO₃ (3×30 mL), 2 M NaHSO₄ (2×30 mL) andbrine. The organic phase was dried with MgSO₄ and evaporated to dryness.The residue was dissolved in THF/LiOH(1M) 2/10 (40 mL) and stirred for 1h after which the pH was adjusted to 2.8 with NaHSO₄. The solution wasextracted with ethyl acetate and the organic phase was dried with MgSO₄;3,6-diaza-(N³-acetyl, N⁶-Boc)-hexanoic acid was collected by evaporationto dryness. Yield 1.8 g (82%).

H¹-NMR DMSO-d₆ δ: 12.31 (s, 1 H, COOH); 6.83 and 6.69 (t, 1 H, BocNH);4.07 and 3.89 (s, 2†H, CH₂CO); 3.30 (m, 2 H, CH₂); 3.09 (m, 2 H, CH₂);2.00 and 1.89 (s, 3 H, CH₃); 1.37 (s,9 H, Boc).

Example 6 Synthesis of PNA Oligomers

The monomers described above were essentially incorporated by theoligomerisation procedure previously described and what has beendisclosed in WO 92/20702. However, the PNA syntheses containing thedonor moieties constituted a special case because the capping step wasomitted in the part of the synthesis including and following the donormonomer. However, to obtain high coupling yields the monomeric moietiesof this part of the synthesis was double coupled. Later experiments haveshown that capping may be used during oligomerisation of donorcontaining molecules. All oligomers were cleaved from the resin by TFMSAand subsequently HPLC purified. The identity was confirmed by massspectroscopy (MALDI-TOF).

PNA 554: calc./found: 5237/5239

PNA 555: calc./found: 5469/5467

PNA 579: calc./found: 5429/5426

PNA 586: calc./found: 5336/5333

PNA 626: calc./found: 6631/6636

PNA 627: calc./found: 6563/6564.

Example 7 Estimation of PNA Concentrations

Concentrations of PNA stock solutions were estimated by UV absorbency at260 nm. The extinction coefficients of the PNA oligomers were calculatedusing the nearest neighbor values for DNA, with Adenine substituted forQ or R. (Because of the length of the PNA oligomers, the errorintroduced by this substitution will be negligible.) In a typicalmeasurement, PNA was dissolved in water and the absorbency at 260 nm wasmeasured after incubation for 5 minutes at 75° C. in order to minimisesecondary structure in the PNA. (Extinction coefficients determined fromroom temperature absorbency measurements tend to be 5-10% lower thanthose measured at the higher temperature). DNA oligomers were purchasedfrom Midland Certified Reagent Company.

Example 8 Formation of Stable PNA/DNA Hybrids: Thermal DenaturationStudies

This example demonstrates the ability of PNAs containing an electronacceptor to hybridize with complementary DNA oligomers.

Samples were prepared consisting of equimolar concentrations of PNA andDNA oligomers (1.0 or 2.0 μM each) in 1.0 mL of 10 mM sodium phosphatebuffer (pH=7.0). The PNA strand will often precipitate upon addition ofthe phosphate, presumably due to complexion of the lysine units by thephosphate. Mixing and addition of the DNA strand results in dissolutionof the PNA. Samples were placed in cuvettes (1.5 mL capacity, 1.0 cmpathlength) and sealed with tape to prevent evaporation of water duringheating/cooling cycles. The absorbency of the samples at 260 nm wasmonitored as a function of temperature for three consecutive runs:heating at 1.0° C./min, and cooling at 0.5° C./min and heating again at0.5° C./min. In two experiments the absorbency was monitored at 330 nm,where the AQ unit absorbs. Due to the low extinction coefficient of theAQ, these experiments were performed at 20 μM each of PNA and DNA.

Data were analysed by exporting as an ASCII file and importing into astandard graphics software. The absorbency was plotted versustemperature for each sample. Melting temperatures (T_(m)) weredetermined as the maxima of plots of the first derivative of absorbencywith respect to temperature, assuming a first order phase transition.T_(m) values given in the text and tables have error values of ±0.5° C.

An initial question to address regarded what could be tolerated on theDNA strand at the central site, i.e. directly across from the AQ in thehybrid. Five variations were considered: (1) An abasic site (X) wasincorporated into the oligomer. This residue replaces the standard DNAbase with a hydrogen atom at carbon-1 of the deoxyribose moiety andwould be expected to provide the most room for accommodation of the AQwithin the duplex. (2)-(5) Each of the four DNA bases: G, A, C and Twere incorporated into the sequence. These DNA oligomers are identifiedas DNA579Z, where Z=X (Abasic), G, A, C, and T, respectively.

DNA: 5′-TCG-CTG-GAA-Z-AAG-GTA-GGA-3′

DNA579Z

PNA: H-Lys-Lys-TCC-TAC-CTT-Y-TTC-CAG-CGA-Gly-NH₂

PNA554: Y=Acetyl

PNAS79: Y=Q1

PNA586: Y=Q2.

The PNA579/DNA579X hybrid melts with high cooperativity at T_(m)=61.4°C. (FIG. 12). There is no hysteresis in the transition indicative ofrapid hybridisation kinetics as previously observed for full-length 1:1PNA-DNA duplexes. The PNA586/DNA579X hybrid melts at 68.3° C.,approximately seven degrees higher than for PNA579. The greaterstabilization for PNA586 could be due to the increased flexibility whicharises from the longer linker connecting the AQ to the PNA backbone. Inboth cases, the T_(m) is significantly higher than when the PNA bearsonly an acetyl group at the central position, indicating that the AQmoiety stabilizes the PNA/DNA hybrid (Table 1).

Monitoring the T_(m) at 330 nm, where only the AQ unit absorbs light,reveals monophasic transitions for both PNA/DNA hybrids (FIG. 12). Thehyperchromicity observed for the PNA586/DNA579X hybrid is nearly twiceas great as that for the PNA579/DNA579X hybrid, consistent with astructure in which the AQ unit is intercalated into the duplex and thatinsertion of the AQ is more favorable for the longer linker.

TABLE 1 Effect of AQ Linker Length on PNA/DNA Hybrid Stability [Duplex]PNA (μM) Wavelength (nm) T_(m) (° C.) % Hypochromicity 554 1.0 260 56.616.5 579 1.0 260 61.4 13.5 586 1.0 260 68.3 15.5

The PNAs form stable hybrids with the DNA579Z oligomers in which Z=G, A,C or T (Table 2). The resulting hybrids are only slightly less stablethan the corresponding duplexes with Z=X.

TABLE 2 Effect of Opposed Base on PNA/DNA Hybrid Stability (T_(m) givenin ° C.) Base PNA554 PNA579 PNA586 X 56.6 61.4 68.3 G 55.6 59.2 65.9 A57.3 59.7 66.6 C 55.8 59.7 67.1 T 56.8 59.5 67.4

Effect of Single Mismatches. PNA555 is analogous to PNA579 with theexception of replacing C with G at position 7. Hybridisation of this PNAwith its abasic DNA complement (DNA555X) yields a duplex which melts at63.1° C. The 2° C. stabilisation relative to PNA579/DNA579X couldreflect greater base stacking in the region of the C-7 (PNA)/G-32 (DNA)base pair. Hibridization of PNA555 with DNA579 yields a duplex whichpossesses a G—G mismatch and melts at 53.4° C. Thus, a single mismatchdepresses T_(m) by 8-10° C.

Effect of PNA Orientation on Hybrid Stability. The preferred orientationof PNA/DNA duplexes is antiparallel, where the PNA N-termini are alignedwith the DNA 3′-termini. If the PNA579 sequence is inverted,hybridization with DNA579 produces a duplex which melts at 49.9° C.Thus, parallel orientation of the PNA and DNA strands results inreduction of the T_(m) by ca. 11° C.

These experiments demonstrate that the DNA recognition properties of thePNA(AQ) conjugates are completely analogous to those of unmodified PNAstrands. While the stability of the resulting duplexes are most likelylower than would be observed for a 19 base pair PNA-DNA hybrid (due tothe lack of base-pairing at the central AQ position), the presence ofthe AQ moiety stabilises the duplex by >4° C. relative to the case whereboth strands contain abasic residues at the central position, indicativeof a strong interaction (stacking) between the AQ and the duplex.

Example 9 Detection of Electron Transfer within PNA/DNA Hybrids byPhotocleavage Assay

This example describes the ability of the PNA containing ananthraquinone (AQ) to initiate photocleavage of the DNA strand within ahybrid duplex by photoinduced electron transfer. Photocleavage of theDNA strand within PNA-AQ/DNA hybrids was studied using radiolabelled DNAand polyacrylamide gel electrophoresis (PAGE). Synthetic DNA oligomerswere labeled at the 5′-OH terminus using [γ-³²P]-ATP and T4polynucleotide kinase according to standard procedures. 5′-endlabelledoligomers were purified by 20% denaturing PAGE and precipitation.Samples were prepared for irradiation by mixing 5.0 μM each of PNA andunlabelled DNA as well as labeled DNA (2000-4000 cpm per sample) in 10mM sodium phosphate buffer (pH=7.0). This ensures that there will be aslight excess of DNA over PNA, minimising contributions fromunhybridised PNA. Samples were heated to 85° C. for 5 min then allowedto cool to room temperature. In cases where the only difference insamples would be the irradiation time, a single sample was prepared bythis procedure and irradiated, with aliquots removed at the desiredtimes.

Irradiation was performed using a Rayonet photoreactor equipped with 8lamps (λ_(max)=350 nm). Samples were placed in microcentrifuge tubes andsuspended parallel to the lamps via a rotating sample holder. While mostof the exciting light is either not incident on the tubes or scatteredby them, the tubes are sufficiently transparent at 330 nm to permitphotocleavage to proceed in reasonable times (less than one hour).

For experiments involving methylene blue, PNA-DNA hybrids were preparedas described above, then methylene blue was added to give 10 or 20 μMconcentration. Samples were irradiated for 15 min using the filtered(λ>600 nm) output of a 150W Hg arc lamp.

For experiments in which D₂O was substituted for water, all of thecomponents (PNA, DNA, buffer, methylene blue where desired) were mixed,then the water was evaporated under vacuum. 20 μl D₂O was added to thesamples and then removed by evaporation. This procedure was repeated,then the samples were finally dissolved in the appropriate amount ofD₂O. An identical procedure was followed using H₂O for control samples.

After irradiation, the salt concentration was increased by addition ofsodium acetate (pH=5.2) to 0.3 M, and magnesium chloride to 10 mM in atotal volume of 50 μL. 100 μL cold ethanol was added and aftervortexing, samples were placed on dry ice for 30 min. followed bycentrifugation at 12,000 g for 30 min. The supernatant was discarded andthe pellet washed with 80% ethanol prior to drying. Samples were eitherthen suspended in denaturing loading buffer or incubated with 100 μLpiperidine (1 M) for 30 min at 90° C. After the piperidine was removedby vacuum evaporation, the DNA was dissolved in 20 μL water which wasthen evaporated. This procedure was performed once more and then the DNAwas suspended in denaturing loading buffer. DNA fragments wereultimately separated on a 20% denaturing polyacrylamide gel and cleavagesites were visualised by autoradiography.

Irradiation of a PNA579/DNA579X duplex with 350 nm light for one hourled to very little spontaneous degradation of the DNA. However, afterpiperidine treatment, strong cleavage is observed at several positions(FIG. 13, Lane 8). Cleavage is not observed for a PNA554/DNA579X hybrid,which lacks an AQ moiety (Lane 4). The main cleavage sites are the threeGG steps as well as the abasic site at the center of the duplex.Previous work has shown that irradiation of DNA-intercalated AQ leads topiperidine-dependent cleavage with high selectivity at the 5′-G of GGdoublets. It has unambiguously been shown in the literature (Breslin, D.T. and Schuster, G. B. J. Am. Chem. Soc. 1996, 118, 2311-2319) that thisGG-selective cleavage of DNA is initiated by photoinduced electrontransfer from the DNA bases to the excited state AQ.

The similar selectivity observed in FIG. 13 suggests that the AQ reactssimilarly with the PNA/DNA hybrid.

For the two GG sites on the 3′ end of the duplex, cleavage at the 5′ Gis favored over that at the 3′ G. Thus, the two GG sites oriented in the3′ direction relative to the AQ exhibit the same 5′-dependent cleavageas observed in duplex DNA. However, cleavage at the GG step on the5′-side of the AQ is distributed evenly between the two sites. The 5′-νs3′-distribution of cleavage at the GG step has been proposed to dependon the angle of rotation between the two guanines as they stack atop oneanother (Sugiyama, H. and Saito, I., J. Am. Chem. Soc. 1996, 118,7063-7068). However, it is important to note that trapping requires notonly a significantly lower oxidation potential at the GG site but also achemical reaction to prevent the electron and hole from recombining.This step involves addition of either water or oxygen to the guanineradical cation, a process which will be influenced by the exposure ofthe base to solvent. It has been noted that the bases in PNA-DNA hybridsare closer to the exterior of the helix than in B-form DNA. NMRErikkson, M.; Nielsen, P. E. Nature Struct. Biol. 1996, 3, 410-413) andx-ray diffraction data (13etts, L.; Josey, J. A.; Veal, J. M.; Jordan,S. R. Science 1995, 270, 1838-1841) indicate that the degree of inter-and intrastrand stacking within PNA-DNA hybrids is highly sequencedependent. Thus there is no reason to expect that the GG cleavage willexhibit the same 5′-preference as observed in duplex DNA. This issupported by the cleavage data on PNA/DNA hybrids disclosed in thisinvention.

Experiments were performed with PNA586/DNA579X which were analogous tothose performed with PNA579. The results for the longer linker aresimilar to the shorter linker: After piperidine treatment, cleavage isobserved at all three GG sites as well as at the abasic site (FIG. 13,Lane 12). The 5′-G is clearly favored for the two sites which are on the3′-side of the abasic site while the two Gs are cleaved approximatelyequally at the other GG site. One notable observation is that thecleavage efficiency for the longer linker connecting the AQ to the PNAbackbone is significantly lower than for the shorter linker.

It is clear that the cleavage is occurring at positions which are farfrom the AQ. In particular, the distal GG step in the 3′ direction isca. 22 Å away from the AQ within the hybrid (based on a rise of 3.6 Åper base pair). Even if the AQ were able to adopt extrahelicalconformations, the short linker connecting it with the backboneprecludes direct reaction between the AQ and the remote G sites. We nextinvestigated the possibility that the AQ was generating a freelydiffusible species. If the PNA and DNA strands are annealed in thepresence of a tenfold excess of unlabeled DNA579X, the cleavage iscompletely eliminated. However, if the annealing is performed in thepresence of an excess of unlabeled, non-complementary DNA, no effect onthe cleavage is observed. This rules out the possible intermediacy of afreely diffusing cleavage agent. Additionally, if the excess unlabeledDNA579X is added after annealing but prior to irradiation, minimalattenuation of the cleavage is observed. This indicates that the PNA/DNAhybrid is thermally stable throughout the irradiation time. Theseexperiments demonstrate that the cleavage arises from excitation of AQmoiety and that the damage is localised at the same duplex wherein thephoton is absorbed.

Selective cleavage at G residues by photonucleases in duplex DNA istypically the result of either electron transfer chemistry or singletoxygen, which can be generated by triplet state molecules. We tested forthe involvement of singlet oxygen by comparing the cleavage efficienciesfor PNA/DNA hybrid in the presence of D₂O with H₂O. (The lifetime ofsinglet oxygen is increased nearly tenfold in the presence of D₂O,leading to significantly enhanced cleavage by singlet oxygengenerators). There is no enhancement of cleavage for the sampleirradiated in D₂O, arguing against a role for singlet oxygen in themechanism. We also studied cleavage of the DNA strand in the PNA/DNAhybrid using a known singlet oxygen generator, methylene blue.Irradiation with visible light (λ>600 nm) results in selective cleavageat the guanine residues with a pattern which is similar to that of theAQ-sensitised cleavage. However, this chemistry is strongly enhancedwhen D₂O is substituted for H₂O.

At first consideration, the similar cleavage patterns observed fordirect irradiation of the AQ and for singlet oxygen generation bymethylene blue would suggest that the quinone-mediated cleavage alsoinvolves singlet oxygen. However, the lack of inhibition by non-specificDNA demonstrates that cleavage is not mediated by freely diffusingsinglet oxygen. The only way that singlet oxygen could be responsiblefor the cleavage is if it is generated by the AQ at the central positionof the duplex, then diffuses one-dimensionally along one of the groovesin either direction until it reacts with a guanine. The differentintensities observed for various Gs would then reflect access of thebase to the singlet oxygen molecule. Such a process should exhibit adistance dependence, however, since the first GG step in the3′-direction would be expected to be a barrier to cleavage at the distalGG step. The equal cleavage observed at these two sites is clearlyinconsistent with such a mechanism. Further evidence against a singletoxygen dependent mechanism comes from the observation thatanthraquinone-2-sulfonate, which is freely diffusing in solution, doesnot induce cleavage of the PNA-DNA hybrid. It is unlikely that thecovalently linked AQ can generate singlet oxygen when the unbound AQcannot, particularly in light of the strong phosphorescence quenching(see Example 10) within the duplex. A further argument against singletoxygen derived cleavage comes from densitometric analysis of the ratioof cleavage at the 5′ and 3′ guanine residues in the distal GG sitetoward the 3′ end of the duplex. For the AQ-initiated cleavage, thisratio is 1.40, whereas for methylene blue it is 0.69. If the sameintermediate were responsible for the DNA damage, then the ratio of5′/3′ cleavage should be the same for AQ and methylene blue.

In light of the preceding discussion, the only explanation for theobserved cleavage is electron transfer in the PNA/DNA duplex. The AQtriplet is quenched by electron transfer from an adjacent base,resulting in the injection of a hole within the duplex. This hole cansubsequently migrate through the helix until it is trapped at a GG site(see FIG. 5). Incorporation into the duplex of a “trap” site, i.e. asite with an even lower oxidation potential than GG, shouldsignificantly alter the cleavage efficiency at the GG sites. The guanineoxidation product, 7,8-dihydro-8-oxoguanine (8-OxoG) was chosen as apotential trap site since its oxidation potential is estimated by Sheuand Foote to be ca. 0.4 V lower than that of guanine (Sheu, C.; Foote,C. S. J. Am. Chem. Soc. 1995, 117, 6439-6442). DNA579T(OxoG) isanalogous to DNA579T except that G-13 is replaced by 8-OxoG. This DNAwas radiolabeled on the 5′-terminus according to standard procedures,then hybridized with PNA579 and irradiated. After piperidine treatment,a significant enhancement is observed in the cleavage at the 8-OxoGposition, while cleavage at the distal GG site is significantlysuppressed relative to the case where a normal G is present at position13. (See FIG. 14, compare lanes 8 and 4). This observation is consistentwith a model in which the excited state AQ accepts an electron from anadjacent base at the intercalation site, injecting a hole into thePNA/DNA helix. This hole migrates along the helix by a series ofdiscrete electron transfers until it reaches a GG site where it can betrapped by reaction with water or oxygen. The (8-OxoG)G site is a moreeffective trap than is a normal GG site, inhibiting migration of thehole to the distal GG site. Significantly, cleavage at the GG site inthe other direction is unaffected by the 8-OxoG.

Example 10 Evidence for Photoinduced Electron Transfer from LowTemperature Phosphorescence Experiments

This example uses phosphorescence spectroscopy to study the reaction ofthe excited state AQ with the PNA/DNA hybrid.

Phosphorescence emission was measured as an indication of electrontransfer from the bases of the hybrid to the photoexcited AQ, sinceelectron transfer quenches this emission. (The quantum yields forphosphorescence are sufficiently low that the samples have to beprepared in a frozen glass matrix in order to be able to detect thephosphorescence. The matrix used was 30% ethylene glycol in 10 mMphosphate buffer, pH=7.0. Stable hybrids are formed even in the presenceof the glycol as evidenced by a suppression of the T_(m) of less than 5°C.) Samples were prepared consisting of AQC(2) (a water soluble AQ), orPNA-AQ alone or with complementary DNA. In each case, the concentrationof AQ (linked or unlinked to PNA) was 5.0 μM, as was the DNAconcentration for the last sample. Samples were prepared in 10 mM sodiumphosphate buffer (pH=7.0) and 30% ethylene glycol, which is required inorder to form a low-temperature glass. For PNA/DNA hybrids, samples wereheated to 85° C. for 5 min then allowed to cool to room temperatureprior to addition of ethylene glycol. Samples (400 μl) were added to NMRtubes, shaken to position the liquid at the bottom of the tube, thenfrozen in liquid nitrogen. Phosphorescence spectra were recorded overthe spectral region of 400-600 nm with excitation at 330 nm. Slits wereset at 5.0 mm on both monochromators to maximise the signal intensity.Spectra were plotted after subtraction of the baseline recorded with afrozen glass lacking AQ and normalisation at 400 nm.

Emission spectra were recorded for three cases: (i) water soluble AQ inthe absence of PNA and DNA, (ii) single-stranded PNA, and (iii) PNA-DNAhybrids. For PNA579, the phosphorescence is quenched by ca. 70% and >90%in the single-strand and hybrid forms, respectively, relative to thefree AQ. Meanwhile, for PNA586, the phosphorescence is quenched by >90%in both the single-stranded and hybrid forms. (Data for PNA586 areplotted in FIG. 15.) These results indicate that there is efficientphotoinduced electron transfer from the bases to the AQ in the hybrid.Moreover, electron transfer is also quite efficient in thesingle-stranded form, indicating the AQ moiety is capable of reactingwith the PNA bases as well as the DNA bases.

Example 11 DNA Recognition by PNA Hairpins

This example describes the use of hybridization to inhibit electrontransfer quenching, demonstrating the ability of PNA/DNA duplexes to actas “bioinsulators”.

In this example, sequence specific recognition of ssDNA by PNA hairpins626 and 627 is demonstrated by thermal denaturation and fluorescencespectroscopy. A similar approach has been reported by Tyagi and Kramer(Tyagi, S.; Kramer, F. R. Nature Biotechnol. 1996, 14, 303-308). In thatstrategy, a DNA hairpin was labeled with an energy donor and acceptor.Hybridization led to decreased energy transfer between the donor andacceptor. The use of PNA in the present invention rather than DNA ispreferred because of the superior hybridization characteristics of PNA,namely the much higher affinity and ionic strength independence of thePNA-DNA recognition. The choice of electron transfer rather than energytransfer in the present invention arises from the fact that fewerrestrictions are placed on the donor and acceptor moieties forphotoinduced electron transfer chemistry. In particular, either thedonor or acceptor can be irradiated and there is no requirement that thedonor absorb light of shorter wavelength than the acceptor.

PNA626 and PNA627 are shown below in their extended and foldedconformations. PNA626 contains an acridine moiety (R₂) which functionsas a light absorber and electron donor plus an anthraquinone moiety (Q₁)which functions as an electron acceptor. In the folded conformation, theacridine and quinone will be placed in close proximity to one another,particularly if both are stacked within the helix, leading to efficientphotoinduced electron transfer, which is detected by quenching of theacridine fluorescence. PNA627 is analogous to PNA626 except it lacks thequinone acceptor. A thymine is included in place of the quinone, leadingto a T—T mismatch in the folded conformation.

PNA626: H-A-T-A-T-Q¹⁻-T-T-G-G-C-T-G-A-T-C-C-A-R₂-T-A-T-A-T-Lys-Lys-NH₂

PNA627: H-A-T-A-T-T-T-T-G-G-C-T-G-A-T-C-C-A-R₂-T-A-T-A-T-Lys-Lys-NH₂

DNA Targets (single stranded, linear)

626A: 5′-T-G-G-A-T-C-A-G-C-C-A-A-3′

626B: 5′-T-G-G-A-T-C-A-G-C-C-T-A-3′

626C: 5′-T-G-G-A-T-C-T-G-C-C-A-A-3′

626D: 5′-A-T-A-T-A-T-T-G-G-A-T-C-A-G-C-C-A-A-T-A-T-A-T-3′.

626A is perfectly complementary to the 12 base sequence separating thequinone from the acridine. 626B and 626C will hybridize with the PNAsbut with single base mismatches. The mismatch for 626B will be locatednear the end of the resulting duplex, yielding a 10 base pair segment,whereas the mismatch for 626C will be located near the center of theresulting duplex, yielding 6 and 5 base pair segments. 626D willhybridize with the full length of the PNAs, placing thymines across fromthe acridine and quinone moieties.

Thermal Denaturation Experiments. Samples were prepared containing 2.5μM PNA in 10 mM sodium phosphate buffer (pH=7.0) with and without 2.5 μMDNA626A. Melting curves were recorded by monitoring the absorbency at260 nm over the temperature range 25-90° C.

FIG. 16 shows the curves for melting of the PNAs in the absence of DNA.The substantial hyperchromicity (>20%) is consistent with a foldedhairpin structure rather than an extended linear conformation. Thetransition midpoint is approximately 55° C. for PNA627. Melting is morecomplex for PNA626 but the transition to the extended conformationoccurs within the range 55-65° C.

FIG. 17 shows the curves for melting of the PNAs in the presence ofDNA626A. The fact that the two curves in this figure are significantlydifferent from those in FIG. 1 demonstrates that the PNA and DNA strandsare interacting with one another. A clear transition is observed at ca.76° C. for both PNAs. This transition is attributed to melting of thedesired PNA/DNA hybrids.

Fluorescence Experiments. Samples were prepared containing 1.0 μM PNA in10 mM sodium phosphate buffer (pH=7.0) with and without 1.0 μM DNA.Samples were heated to 90° C. for 5 min, then cooled to room temperatureover a period of 90 minutes. Fluorescence emission spectra were recordedover the range 430-600 nm with excitation at 417 nm. 2.5 mm slits, 1.0nm increment and 0.5 sec integration time were used for data collection.After collection, spectra were integrated to give the data shown below.

TABLE 3 Effect of Hybridization on ET Quenching of Acridine FluorescenceSample PNA DNA Raw Data Normalized Data* 1 626 None 3.35 1.00 2 626 626A12.0 3.58 3 626 626B 11.0 3.28 4 626 626C 9.98 2.98 5 626 626D 25.1 7.496 627 None 12.1 1.00 7 627 626A 16.5 1.37 8 627 626B 16.4 1.36 9 627626C 15.2 1.26 10  627 626D 32.3 2.67 *Data for each PNA were normalizedto the values for the samples lacking DNA (1 and 6).

Effect of Quinone in PNA Hairpin. Comparing samples 1 and 6, thefluorescence is 3.6 times lower when the quinone is present, consistentwith quenching of the acridine fluorescence by electron transfer to theadjacent quinone. The lack of complete quenching is most likely due tothe fact that the electron transfer reaction is not very favorableenergetically and the lifetime of the acridine is probably fairly short(a few nanoseconds). An additional factor, indicated by the broadhairpin melting curves, is that the acridine and quinone likely havemultiple conformations (e.g. intercalated or extrahelical). Theincomplete quenching could arise from a population of hairpins in whichonly one of the two chromophores is actually intercalated at the time ofexcitation.

Effect of Hybridization. In the presence of complementary DNA, thehairpin is disrupted and a PNA/DNA hybrid is formed. In the hybrid, theacridine and anthraquinone are separated by 12 base pairs instead ofbeing in contact in the hairpin. This results in a substantial increasein the fluorescence (compare samples 1 and 2). The increase is muchgreater for PNA626 than for 627, indicating that most of the enhancedfluorescence results from retarded electron transfer rather than fromthe change in the environment of the acridine fluorophore due tohybridization with the DNA strand. Note that the fluorescence is stillgreater for PNA627, suggesting that there is still some electrontransfer quenching occurring over 12 base pairs within the hybrid.

Effect of Single Base Mismatches. A single mismatch near the end of therecognition site results in a 12% decrease in fluorescence enhancementwhile a mismatch near the center of the recognition site results in a23% decrease in fluorescence enhancement. As expected, the centralmismatch results in shorter (and, therefore, less stable) hybridregions, leading to less fluorescence enhancement.

Effect of full-length Hybridization. The largest enhancements resultsfrom hybridization with a full length DNA complement (DNA626D, seeSamples 5 and 10). This result emphasizes the sensitivity of theacridine fluorescence quantum yield to its environment: the emission issignificantly greater when the acridine is part of a duplex as opposedto in a single strand. (The results for PNA627 also indicate that theacridine fluorescence quantum yield is greater for a PNA-DNA versus aPNA-PNA hybrid. The fluorescence increases by 2.67 in the presence ofDNA.) The combination of this effect with the relieved electron transferresults in the large enhancement of fluorescence.

Assessment. The donor-acceptor PNA hairpins appear to work as designedand significant enhancements of fluorescence arise from hybridizationwith all four DNA oligomers. In each case, the fluorescence was enhanced2.4-2.8 times more for PNA626 than for PNA627, demonstrating theimportance of the electron acceptor in the system. The experiments wereperformed on samples containing 1 nanomole each of PNA and DNA target;this could easily be decreased by a factor of 10.

Example 12 PNA Hairpin using a different Electron Donor

In this experiment, denomination is corresponding to example 11. N meansthe monomer containing an ANI residue. The N-monomer is shown in FIG.18.

PNA 800: H-ATA-TQT-TGG-CTG-ATC-CAN-TAT-AT-LYS-Lys-NH2

PNA 801: H-ATA-TQX-TGG-CTG-ATC-CAN-XAT-AT-LYS-Lys-NH2

PNA 802: H-ATA-TTT-TGG-CTG-ATC-CAN-TAT-AT-LYS-Lys-NH2.

In PNA 800 T's are opposite Q and N. In PNA 801 a no-base X is oppositethe Q and N supposed to give more room for the bulky chromophores. In802 only ANI is placed.

DNAs: (single stranded, linear)

DNA A: 3′-ACC-GAC-TAG-GT-5′

DNA D: 3′-TAT-ATA-ACC-GAC-TAG-GTT-ATA-TA-5′.

Analogous Tm experiments as in the acridine/Q system was performed, andhairpin formation of individual PNA oligomers, and hybrid formation foreach PNA oligomer to both DNA A and D was shown. Fluorescenceexperiments were then performed as described below and quantificationwas as follows:

Sample Sample ingredients Fluorescence Normalised 1. 1 μM PNA 800  3.6 ×10⁶ 1 2. 1 μM PNA 800 + 1 μM DNA A 11.8 × 10⁶ 3.6 3. 1 μM PNA 800 + 1 μMDNA D 20.3 × 10⁶ 5.6 4. 1 μM PNA 801  2.4 × 10⁶ 1 5. 1 μM PNA 801 + 1 μMDNA A 11.2 × 10⁶ 4.6 6. 1 μM PNA 801 + 1 μM DNA D 12.9 × 10⁶ 5.4 7. 1 μMPNA 802 13.6 × 10⁶ 1 8. 1 μM PNA 802 + 1 μM DNA A 15.0 × 10⁶ 1.1 9. 1 μMPNA 802 + 1 μM DNA D 14.7 × 10⁶ 1.1

PNA 802 has no Q monomer and there is therefore no difference in thefluorescence difference between the hairpin and the unfolded hairpin,PNA-DNA duplex. In both PNA 800 and 801 the quinone quenches thefluorescence when the hairpin is formed. This shown that excitation ofANI results in electron transfer to the quinone, thus no radiative eventhappens. When either the short or the long DNA is present the hairpin isunfolded and the quenching by the quinone is significantly reducedleading to fluorescence by the ANI chromophore. This experiment showsthat electron transfer is not immediate in PNA/DNA and that theintervening bases pairs exerts this property.

a) N-monomer-Synthesis

Benzyl N-(4-amino-1,8-napthalimido)glycinate

To a suspension of 2.1 g (10 mmol) of 4-amino-1,8-napthalimide andpotassium carbonate (4.14 g, 30 mmol) in DMF (50 ml) was stirred at 40°C. for 0.5 h . The suspension was cooled to room temperature and (12mmol, 1.8 ml) benzyl bromoacetate was added in one portion. Thesuspension was stirred over night. To the reaction was added 10 g ofcelite and filtered through 1 cm of celite on a filter. The solvent wasremoved under reduced pressure and the residue was crystallised fromethylacetate.

Yield 3.1 g (89%); ¹H NMR (DMSO-d₆): δ 4.84 (s, 2 H, CH₂CO); 5.19 (s, 2H, CH₂O); 6.87 (d, 1 H, H-3); 7.37 (m, 5 H, benzyl); 7 56 (s, 2 H, NH₂);7.68 (t, 1 H, H-6); 8.21 (d, 1 H, H-5); 8.46 (dd, 1 H, H-2); 8.67 (dd, 1H, H-7).

N-(4-amino-1,8-napthalimido)glycine

To a solution of benzyl N-(4-amino-1,8-napthalimido)glycinate (2.5 g,7.2 mmol) in THF (20 ml) was added lithium hydroxide (30 ml, 1 M) andstirred for 3 h. The THF was removed under reduced pressure and pH wasadjusted to 2.8 and the precipitated free acid was collected byfiltration and washed with water and dried.

Yield 1.6 g (86%). ¹H NMR (DMSO-d₆): δ 4.66 (s, 2 H, CH₂CO); 6.86 (d, 1H, H-3); 7 52 (s, 2 H, NH₂); 7.67 (t, 1 H, H-6); 8.20 (d, 1 H, H-5);8.45 (dd, 1 H, H-2); 8.66 (dd, 1 H, H-7).

N,N-(2-(N-(4-amino-1,8-napthalimido)glycinyl))-(2Boc-aminoethyl))glycine

N-(4-amino-1,8-napthalimido)glycine (1.5 g, 5.8 mmol), DCC (1.6 g, 6.2mmol) and DhbtOH (1.0 g, 6.2 mmol) was mixed and stirred for 10 min inDMF (35 ml). Then methyl N-(-2-Boc-aminoethyl)glycinate (1.5 g, 6.5mmol) was added and the reaction was stirred for additional 4 h. To thereaction mixture dichloromethane (70 ml) was added after which themixture was cooled to 0° C. and filtered. The filtrate was extractedwith sodium bicarbonate (3×35 ml, 0.5 M), sodium bisulphate (2×35 ml 2M) and brine. The organic phase was dried with magnesium sulphate andevaporated to dryness. The residue was dissolved in tetrahydrofurane (20ml) and lithium hydroxide (40 ml of 1 M) was added. The reaction mixturewas stirred for 3 h. Tetrahydrofurane was removed under reduced pressureand the solution was filtrated and pH of the filtrate was adjusted to2.8 which precipitated the title compound. The precipitate wasrecrystallised from ethylacetate.

Yield 2.1 g (72%); ¹H NMR (DMSO-d₆): δ 1.37 (d, 9H, Boc); 3.04 (m, 2 H,CH₂); 3.27 (m, 2H, CH₂); 4.02 (d, 2 H, CH₂CO); 4.80 (d, 2H, CH₂CO); 6.86(m, 1 H, H-3); 6.95 (m, 1H, NH); 7 52 (s, 2 H, NH₂); 7.63 (m, 1 H, H-6);8.14 (m, 1 H, H-5); 8.36 (m, 1 H, H-2); 8.64 (m, 1 H, H-7).

b) PNA Synthesis

The three PNA oligomers were synthesised accordingly to the standardschemes. The oligomers were HPLC purified and the identity was checkedby mass spectroscopy (MALDI-TOF).

Calcd/found PNA 800: 6634/6643 PNA 801: 6386/6384 PNA 802: 6576/6576

c) Fluorescence Measurements

DNA oligomers were purchased from Midland Certified Reagent Company,purified by gel filtration and characterised by MALDI-TOF massspectrometry. The extinction coefficients of DNA oligomers werecalculated using the nearest-neighbor values: for DNA A, ε₂₆₀=108,200M⁻¹cm⁻¹; DNA D, ε₂₆₀=238,200 M⁻¹cm⁻¹. Similarly, PNA oligomersextinction coefficients were determined using DNA values, with adeninesubstituted for 4-amino-1,8-naphthalimide or anthraquinone: PNA 800,ε₂₆₀=232,900 M⁻¹cm⁻¹; PNA 801, ε₂₆₀=214,700 M⁻¹cm⁻¹; PNA 802,ε₂₆₀=228,900 M⁻¹cm⁻¹. The samples for all experiments were prepared in a10 mM sodium phosphate buffer at pH=7.0. Fluorescence spectra wererecorded with Cary 1E (UV-Vis) and SPEX 1681 FLUOROLOG. For PNAhairpins, samples were prepared consisting of 1.0 μM of PNA oligomer in1.0 mL of 10 mM sodium phosphate (pH 7.0). In the case of PNA/DNAhybrids, equimolar concentration of PNA and DNA oligomers (1.0μ each)were used. Samples were placed in cuvettes (1.5 mL capacity, 1.0 cmpathlength) and sealed with Teflon tape to prevent evaporation of waterduring heating/cooling cycles. The same samples were heated to 90° C.for 5 min and then slowly cooled to RT to assure complete hybridisation.Samples were placed in a fluorescence cuvette (from Hellma: 1.5 mLcapacity, 1.0 cm pathlength) and excited at 450 nm. The fluorescenceemission was monitored from 460 to 700 nm at the rate of 1 nm/sec withthe following slit sizes: ex=2.5 mm and em=0.5 mm.

What is claimed is:
 1. A method for determining a nucleic acid in asample comprising binding to the nucleic acid a probe molecule having apolymeric backbone different from the natural sugar phosphate backbonevia base mediated hydrogen bonding, wherein an electron acceptor or anelectron donor or an electron acceptor and an electron donor iscovalently bound to said probe molecule at a non-terminal subunit ofsaid polymeric backbone not bearing a nucleobase, inducing an electrontransfer from said electron donor or to said elector acceptor anddetermining the occurrence of the electronic transfer as a measure ofsaid nucleic acid.
 2. A method according to claim 1, characterized inthat the electronic transfer is induced by photo inducement.
 3. A methodaccording to claim 1, characterized in that said electron donor is anucleobase.
 4. A method according to claim 1, characterized in that saidelectron donor is bound to said probe molecule.
 5. A method according toclaim 1, characterized in that said electron donor is bound to a furtherprobe molecule bound to said nucleic acid.
 6. A method according toclaim 1, characterized in that said electron acceptor is bound to saidprobe molecule at a position which is different from the last nucleobasebearing subunit of said probe molecule.
 7. A method according to claim1, characterized in that said occurrence of electron transfer isdetermined by analyzing changes in the electronic or photometric statusof said electron acceptor.
 8. A method according to claim 1,characterized in that said occurrence of electron transfer is determinedby analyzing changes in the electronic or photometric status of saidelectron donor.
 9. A method according to claim 1, characterized in thatsaid occurrence of electron transfer is determined by detecting a changein the molecular structure of said nucleic acid compared to thestructure without the inducement of said electron transfer.
 10. A methodaccording to claim 1, characterized in that the electron donor is aguanine base.
 11. A method according to claim 1, characterized in thatthe electron donor is an electrode.
 12. A method according to claim 1,characterized in that the probe molecule is bound to a solid phase. 13.A method according to claim 10, characterized in that said change is thecleavage of a nucleobase from said backbone.
 14. A method according toclaim 1, characterized in that said backbone comprises at least onepeptide bond.
 15. The method of claim 1, wherein said electron acceptoris a molecule of the general formula IIIa or IIIb,

wherein X, Y, Z, Q, V, and W are independently selected from the atomsC, N, S, and O; X, Y, Z, Q, V, and W are connected by either single ordouble bonds; R₁-R₆ are independently selected from the group of —H,—O⁻, —OH, —OR′, —SH, —SR′, —NH₂, —NO₂, —SO₃ ⁻, —SO₂ ⁻, —CN, —PO₃ ²⁻,—PO₂ ⁻, —COOH, —CO—R′, —COOR′, —CS—R′, —CSO—R′, —COO⁻, —N═N—, halogen,—NHR′, —N(R′R″), hydrocarbyl, and heterocycle; R′ and R″ areindependently selected from the same groups as R₁-R₆; wherein at leastone of X, Y, Z, Q, V, and W can together with any one of R₁-R₆ also be—CO⁻, —SO⁻ or —SO₂ ⁻; and at least one of R₁-R₆ can be a single ordouble bond.
 16. The method of claim 1 wherein the polymeric backbonedifferent from the natural sugar phosphate backbone is a peptide nucleicacid (PNA).
 17. The method of claim 1 wherein one of the electron donoror electron acceptor is not a naturally occurring nucleobase.
 18. Themethod of claim 1 wherein both the electron donor and electron acceptorare not naturally occurring nucleobases.
 19. The method of claim 9wherein said change is the cleavage of the backbone of the nucleic acid.